Isolation and characterization of a novel pythium omega 3 desaturase with specificity to all omega 6 fatty acids longer than 18 carbon chains

ABSTRACT

The present invention relates to a polynucleotide encoding an omega 3 (ω-3) desaturase from  Pythium irregulare  with specificity to long chain polyunsaturated omega 6 (ω-6) fatty acids as well as a vector containing said polynucleotide, and a host cell containing the vector or the polynucleotide. Moreover, the present invention pertains to a polypeptide encoded by the said polynucleotide, antibodies against the polypeptide as well as a method for the manufacture of the polypeptide. Further, encompassed by the present invention are transgenic non-human organisms. Finally, the present invention relates to methods for the manufacture of compounds and oil- fatty acid- or lipid-containing compositions.

The present invention relates to a polynucleotide encoding an omega 3(ω-3) desaturase from Pythium irregulare with specificity to long chainpolyunsaturated omega 6 (ω-6) fatty acids as well as a vector containingsaid polynucleotide, and a host cell containing the vector or thepolynucleotide. Moreover, the present invention pertains to apolypeptide encoded by the said polynucleotide, antibodies against thepolypeptide as well as a method for the manufacture of the polypeptide.Further, encompassed by the present invention are transgenic non-humanorganisms. Finally, the present invention relates to methods for themanufacture of compounds and oil- fatty acid- or lipid-containingcompositions.

Fatty acids and triacylglycerides have a multiplicity of applications inthe food industry, in animal nutrition, in cosmetics and thepharmacological sector. Depending on whether they are free saturated orunsaturated fatty acids or else triacylglycerides with an elevatedcontent of saturated or unsaturated fatty acids, they are suitable forvarious different applications.

Polyunsaturated long-chain ω-3-fatty acids such as eicosapentaenoic acid(=EPA, C20:5^(Δ5,8,11,14,17)), ω-3 eicostetraenic acid (=ETA,C20:4^(Δ8,11,14,17)) or docosahexaenoic acid (=DHA,C22:6^(Δ4,7,10,13,16,19)) are important components of human nutritionowing to their various roles in health aspects, including thedevelopment of the child brain, the functionality of the eyes, thesynthesis of hormones and other signal substances, and the prevention ofcardiovascular disorders, cancer and diabetes (Poulos, A Lipids 30:1-14,1995; Horrocks, L A and Yeo Y K Pharmacol Res 40:211-225, 1999). Thereis, therefore, a need for the production of polyunsaturated long-chainfatty acids.

Owing to the present-day composition of human food, an addition ofpolyunsaturated ω-3-fatty acids, which are preferentially found in fishoils, to the food is particularly important. Thus, for example,polyunsaturated fatty acids such as DHA or EPA are added to infantformula to improve the nutritional value. The unsaturated fatty acid DHAis supposed to have a positive effect on the development and maintenanceof brain functions.

In the following, polyunsaturated fatty acids are sometimes referred toas PUFA, PUFAs, LCPUFA or LCPUFAs (poly unsaturated fatty acids, PUFA,long chain poly unsaturated fatty acids, LCPUFA).

The various fatty acids and triglycerides are mainly obtained frommicroorganisms such as Mortierella or Schizochytrium or fromoil-producing plants such as soybeans, oilseed rape, algae such asCrypthecodinium or Phaeodactylum and others, being obtained, as a rule,in the form of their triacylglycerides (=triglycerides=triglycerols).However, they can also be obtained from animals, for example, fish. Thefree fatty acids are, advantageously, prepared by hydrolysis. Verylong-chain polyunsaturated fatty acids such as DHA, EPA, arachidonicacid (=ARA, C20:4^(Δ5,8,11,14)), dihomo-γ-linolenic acid (=DGLA,C20:3^(Δ8,11,14)) or docosapentaenoic acid (DPA, C22:5^(Δ7,10,13,16,19))are not synthesized in plants, for example in oil crops such as oilseedrape, soybeans, sunflowers and safflower. Conventional natural sourcesof these fatty acids are fish such as herring, salmon, sardine, redfish,eel, carp, trout, halibut, mackerel, zander or tuna, or algae. Dependingon the intended use, oils with saturated or unsaturated fatty acids arepreferred. In human nutrition, for example, lipids with unsaturatedfatty acids, specifically polyunsaturated fatty acids, are preferred.The polyunsaturated ω-3-fatty acids are said to have a positive effecton the cholesterol level in the blood and thus on the possibility ofpreventing heart disease. The risk of heart disease, stroke orhypertension can be reduced markedly by adding these ω-3-fatty acids tothe food. Also, ω-3-fatty acids have a positive effect on inflammatory,specifically on chronically inflammatory, processes in association withimmunological diseases such as rheumatoid arthritis. They are,therefore, added to foodstuffs, specifically to dietetic foodstuffs, orare employed in medicaments. ω-6-fatty acids such as arachidonic acidtend to have an adverse effect on these disorders in connection withthese rheumatic diseases on account of our usual dietary intake.

ω-3- and ω-6-fatty acids are precursors of tissue hormones, known aseicosanoids, such as the prostaglandins. The prostaglandins which arederived from dihomo-γ-linolenic acid, arachidonic acid andeicosapentaenoic acid, and of the thromoxanes and leukotrienes, whichare derived from arachidonic acid and eicosapentaenoic acid. Eicosanoids(known as the PG₂ series) which are formed from the ω-6-fatty acidsgenerally promote inflammatory reactions, while eicosanoids (known asthe PG₃ series) from ω-3-fatty acids have little or no proinflammatoryeffect. Therefore, food having a high proportion of ω-3-fatty acid has apositive effect on human health.

Owing to their positive characteristics, there has been no lack ofattempts in the past to make available genes which are involved in thesynthesis of fatty acids or triglycerides for the production of oils invarious organisms with a modified content of unsaturated fatty acids.Thus, WO 91/13972 and its US equivalent describe a Δ9-desaturase. WO93/11245 claims a Δ15-desaturase and WO 94/11516 a Δ12-desaturase.Further desaturases are described, for example, in EP A 0 550 162, WO94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EPA 0 794 250, Stukeyet al., J. Biol. Chem., 265, 1990: 20144-20149, Wada et al., Nature 347,1990: 200-203 or Huang et al., Lipids 34, 1999: 649-659. However, thebiochemical characterization of the various desaturases has beeninsufficient to date since the enzymes, being membrane-bound proteins,present great difficulty in their isolation and characterization (McKeonet al., Methods in Enzymol. 71, 1981: 12141-12147, Wang et al., PlantPhysiol. Biochem., 26, 1988: 777-792). As a rule, membrane-bounddesaturases are characterized by being introduced into a suitableorganism which is subsequently analyzed for enzyme activity by analyzingthe starting materials and the products. Δ6-Desaturases are described inWO 93/06712, U.S. Pat. No. 5,614,393, U.S. Pat. No. 5,614,393, WO96/21022, WO 00/21557 and WO 99/27111, and also the application for theproduction in transgenic organisms is described in WO 98/46763, WO98/46764 and WO 98/46765. Here, the expression of various desaturases isalso described and claimed in WO 99/64616 or WO 98/46776, as is theformation of polyunsaturated fatty acids. As regards the expressionefficacy of desaturases and its effect on the formation ofpolyunsaturated fatty acids, it must be noted that the expression of asingle desaturase as described to date has only resulted in low contentsof unsaturated fatty acids/lipids such as, for example, γ-linolenic acidand stearidonic acid. Furthermore, mixtures of ω-3- and ω-6-fatty acidsare usually obtained.

Especially suitable microorganisms for the production of PUFAs aremicroorganisms including microalgae such as Phaeodactylum tricornutum,Porphiridium species, Thraustochytrium species, Schizochytrium speciesor Crypthecodinium species, ciliates such as Stylonychia or Colpidium,fungi such as Mortierella, Entomophthora or Mucor and/or mosses such asPhyscomitrella, Ceratodon and Marchantia (R. Vazhappilly & F. Chen(1998) Botanica Marina 41: 553-558; K. Totani & K. Oba (1987) Lipids 22:1060-1062; M. Akimoto et al. (1998) Appl. Biochemistry and Biotechnology73: 269-278). Strain selection has resulted in the development of anumber of mutant strains of the microorganisms in question which producea series of desirable compounds including PUFAs. However, the mutationand selection of strains with an improved production of a particularmolecule such as the polyunsaturated fatty acids is a time-consuming anddifficult process. Thus, recombinant methods are preferred whereverpossible. However, only limited amounts of the desired polyunsaturatedfatty acids such as ETA, DHA or EPA can be produced with the aid of theabovementioned microorganisms; where they are generally obtained asfatty acid mixtures of, for example, ETA, EPA and DHA, depending on themicroorganism used.

A variety of synthetic pathways is being discussed for the synthesis ofthe polyunsaturated fatty acids, eicosapentaenoic acid anddocosahexaenoic acid. EPA or DHA are produced in numerous marinebacteria such as Vibrio sp. or Shewanella sp. via the so-calledpolyketide pathway (Yu, R. et al. Lipids 35:1061-1064, 2000; Takeyama,H. et al. Microbiology 143:2725-2731, 1197)).

An alternative strategy is the alternating activity of desaturases andelongases (Zank, T. K. et al. Plant Journal 31:255-268, 2002;Sakuradani, E. et al. Gene 238:445-453, 1999). A modification of theabove-described pathway by Δ6-desaturase, Δ6-elongase, Δ5-desaturase,Δ5-elongase and Δ4-desaturase is the Sprecher pathway (Sprecher 2000,Biochim. Biophys. Acta 1486:219-231) in mammals. Instead of theΔ4-desaturation, a further elongation step is effected here to give C₂₄,followed by a further Δ6-desaturation and finally β-oxidation to givethe C₂₂ chain length. What is known as the Sprecher pathway is, however,not suitable for the production in plants and microorganisms since theregulatory mechanisms are not known.

Depending on their desaturation pattern, the polyunsaturated fatty acidscan be divided into two large classes, viz. ω-6- or ω-3-fatty acids,which differ with regard to their metabolic and functional activities.

The starting material for the ω-6-metabolic pathway is the fatty acidlinoleic acid (18:2^(Δ9,12)) while the ω-3-pathway proceeds vialinolenic acid (18:3^(Δ9,12,15)). Linolenic acid is formed by theactivity of an ω-3-desaturase (Tocher et al. 1998, Prog. Lipid Res. 37,73-117; Domergue et al. 2002, Eur. J. Biochem. 269, 4105-4113).

Mammals, and thus also humans, have no corresponding desaturase activity(Δ12- and ω-3-desaturase) and must take up these fatty acids (essentialfatty acids) via the food. Starting with these precursors, thephysiologically important polyunsaturated fatty acids arachidonic acid(=ARA, 20:4^(Δ5,8,11,14)), an ω-6-fatty acid and the two ω-3-fatty acidseicosapentaenoic acid (=EPA, 20:5^(Δ5,8,11,14,17)) and docosa-hexaenoicacid (DHA, 22:6^(Δ4,7,10,13,17,19)) are synthesized via the sequence ofdesaturase and elongase reactions. The application of ω-3-fatty acidsshows the therapeutic activity described above in the treatment ofcardiovascular diseases (Shimikawa 2001, World Rev. Nutr. Diet. 88,100-108), inflammations (Calder 2002, Proc. Nutr. Soc. 61, 345-358) andarthritis (Cleland and James 2000, J. Rheumatol. 27, 2305-2307).

From the angle of nutritional physiology, it is, therefore, important toachieve a shift between the ω-6-synthetic pathway and the ω-3-syntheticpathway (see FIG. 1) in the synthesis of polyunsaturated fatty acids sothat more ω-3-fatty acids are produced. The enzymatic activities ofvarious ω-3-desaturases which desaturate C_(18:2)-, C_(22:4)- orC_(22:5)-fatty acids have been described in the literature (see FIG. 1).However, none of the desaturases whose biochemistry has been describedconverts a broad range of substrates of the ω-6-synthetic pathway intothe corresponding fatty acids of the ω-3-synthetic pathway.

There is therefore still a great demand for an ω-3-desaturase which issuitable for the production of ω-3-polyunsaturated fatty acids. All theknown plant and cyanobacterial ω-3-desaturases desaturate C18-fattyacids with linoleic acid as the substrate, but cannot desaturate C20- orC22-fatty acids.

An ω-3-desaturase which can desaturate C20-polyunsaturated fatty acidsis known from the fungus Saprolegnia dicilina (Pereira et al. 2003,Biochem. J. 2003 Dez, manuscript BJ20031319). However, it isdisadvantageous that this ω-3-desaturase cannot desaturate C18- orC22-PUFAs, such as the important fatty acids C18:2-, C22:4- orC22:5-fatty acids of the ω-6-synthetic pathway. A further disadvantageof this enzyme is that it cannot desaturate fatty acids which are boundto phospholipids. Only the CoA-fatty acid esters are converted.Recently, other ω-3-desaturases have been described with a pivotalsubstrate specificity for ARA, DGLA and Docosatetraenoic acid(=DTA^(Δ8,11,14,17) (WO2005/083053).

To make possible the fortification of food and/or of feed withpolyunsaturated ω-3-fatty acids, there is still a great need for asimple, inexpensive process for the production of each of theaforementioned long chain polyunsaturated fatty acids, especially ineukaryotic systems.

The technical problem underlying the present invention, thus, could beseen as the provision of means and methods which allow the synthesis ofLCPUFAs and which allow a shift from the ω-6-synthetic pathway to theω-3-synthetic pathway in order to manufacture polyunsaturated fattyacids and derivatives thereof. The technical problem has been solved bythe embodiments characterized below and in the accompanying claims.

Accordingly, the present invention relates to a polynucleotidecomprising a nucleic acid sequences selected from the group consistingof:

-   -   (a) a nucleic acid sequence as shown in SEQ ID NO: 1 or 23;    -   (b) a nucleic acid sequence encoding a polypeptide having an        amino acid sequence as shown in SEQ ID NO: 2 or 24;    -   (c) a nucleic acid sequence which is at least 70% identical to        the nucleic acid sequence of (a) or (b), wherein said nucleic        acid sequence encodes a polypeptide having ω-3 desaturase        activity;    -   (d) a nucleic acid sequence being a fragment of any one of (a)        to (c), wherein said fragment encodes a polypeptide having ω-3        desaturase activity; and    -   (e) a nucleic acid sequence encoding a polypeptide having ω-3        desaturase activity, wherein said polypeptide comprises a        polypeptide pattern as shown in a sequence selected from the        group consisting of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22,        37, 38, 39, 40, 41, 42, 43, 44 and 45.

The term “polynucleotide” as used in accordance with the presentinvention relates to a polynucleotide comprising a nucleic acid sequencewhich encodes a polypeptide having ω-3 desaturase activity, i.e. beingcapable of converting a ω-6 PUFA into its corresponding ω-3 PUFA. Morepreferably, the polypeptide encoded by the polynucleotide of the presentinvention shall be capable of introducing a double bond on theω-3-position into a ω-6 PUFA. The ω-6 PUFA is, preferably, an LCPUFA,more preferably, a C20- or C22-PUFA. C20- and C22-PUFAs are alsoreferred to as LCPUFAs herein below. Most preferably, the polynucleotideof the present invention encodes a polypeptide which is capable ofconverting ω-6 DPA into DHA. Suitable assays for measuring theactivities mentioned before are described in the accompanying Examplesor in WO2005/083053. A polynucleotide encoding a polypeptide having theaforementioned biological activity has been obtained in accordance withthe present invention from Pythium irregulare. Thus, the polynucleotide,preferably, comprises the nucleic acid sequence shown in SEQ ID NO: 1 or23 encoding the polypeptide having an amino acid sequence as shown inSEQ ID NO: 2 or 24, respectively. The two polypeptides shall representisoforms of the ω3-desaturase of the present invention. It is to beunderstood that a polypeptide having an amino acid sequence as shown inSEQ ID NO: 2 or 24 may be also encoded due to the degenerated geneticcode by other polynucleotides as well.

Moreover, the term “polynucleotide” as used in accordance with thepresent invention further encompasses variants of the aforementionedspecific polynucleotides. Said variants may represent orthologs,paralogs or other homologs of the polynucleotide of the presentinvention. Homolgous polynucleotides are, preferably, polynucleotidescomprise sequences as shown in any one of SEQ ID NO: 6, 7, 9, 11, 13,30, 33 or 35 or those which encode polypeptides comprising amino acidsequences as shown in any one of SEQ ID NOs: 8, 10, 12, 14, 31, 34 or36.

The polynucleotide variants, preferably, also comprise a nucleic acidsequence characterized in that the sequence can be derived from theaforementioned specific nucleic acid sequences shown in SEQ ID NO: 1 or23 or in any one of SEQ ID NOs: 6, 7, 9, 11, 13, 30, 33 or 35 by atleast one nucleotide substitution, addition and/or deletion whereby thevariant nucleic acid sequence shall still encode a polypeptide havingω-3 desaturase activity as specified above. Variants also encompasspolynucleotides comprising a nucleic acid sequence which is capable ofhybridizing to the aforementioned specific nucleic acid sequences,preferably, under stringent hybridization conditions. These stringentconditions are known to the skilled worker and can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. A preferred example for stringent hybridization conditionsare hybridization conditions in 6× sodium chloride/sodium citrate (=SSC)at approximately 45° C., followed by one or more wash steps in 0.2×SSC,0.1% SDS at 50 to 65° C. The skilled worker knows that thesehybridization conditions differ depending on the type of nucleic acidand, for example when organic solvents are present, with regard to thetemperature and concentration of the buffer. For example, under“standard hybridization conditions” the temperature differs depending onthe type of nucleic acid between 42° C. and 58° C. in aqueous bufferwith a concentration of 0.1 to 5×SSC (pH 7.2). If organic solvent ispresent in the abovementioned buffer, for example 50% formamide, thetemperature under standard conditions is approximately 42° C. Thehybridization conditions for DNA:DNA hybrids are, preferably, 0.1×SSCand 20° C. to 45° C., preferably between 30° C. and 45° C. Thehybridization conditions for DNA:RNA hybrids are, preferably, 0.1×SSCand 30° C. to 55° C., preferably between 45° C. and 55° C. Theabovementioned hybridization temperatures are determined for example fora nucleic acid with approximately 100 bp (=base pairs) in length and aG+C content of 50% in the absence of formamide. The skilled worker knowshow to determine the hybridization conditions required by referring totextbooks such as the textbook mentioned above, or the followingtextbooks: Sambrook et al., “Molecular Cloning”, Cold Spring HarborLaboratory, 1989; Hames and Higgins (Ed.) 1985, “Nucleic AcidsHybridization: A Practical Approach”, IRL Press at Oxford UniversityPress, Oxford; Brown (Ed.) 1991, “Essential Molecular Biology: APractical Approach”, IRL Press at Oxford University Press, Oxford.Alternatively, polynucleotide variants are obtainable by PCR-basedtechniques such as mixed oligonucleotide primer-based amplification ofDNA, i.e. using degenerated primers against conserved domains of thepolypeptides of the present invention. Conserved domains of thepolypeptide of the present invention may be identified by a sequencecomparison of the nucleic acid sequence of the polynucleotide or theamino acid sequence of the polypeptide of the present invention withother ω-3 desaturase sequences (see, e.g., FIG. 3). Oligonucleotidessuitable as PCR primers as well as suitable PCR conditions are describedin the accompanying Examples. As a template, DNA or cDNA from bacteria,fungi, plants or animals may be used. Further, variants includepolynucleotides comprising nucleic acid sequences which are at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98% or at least 99% identical to the nucleic acidsequences shown in SEQ ID NO: 1 or 23 retaining ω-3 desaturase activity.Moreover, also encompassed are polynucleotides which comprise nucleicacid sequences encoding amino acid sequences which are at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98% or at least 99% identical to the amino acid sequences shown inSEQ ID NO: 2 or 24 or an amino acid sequence as shown in any one of SEQID NOs: 8, 10, 12, 14, 31, 34 or 36 wherein the polypeptide comprisingthe amino acid sequence retains ω-3 desaturase activity. The percentidentity values are, preferably, calculated over the entire amino acidor nucleic acid sequence region. A series of programs based on a varietyof algorithms is available to the skilled worker for comparing differentsequences. In this context, the algorithms of Needleman and Wunsch orSmith and Waterman give particularly reliable results. To carry out thesequence alignments, the program PileUp (J. Mol. Evolution., 25,351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or the programsGap and BestFit (Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970))and Smith and Waterman (Adv. Appl. Math. 2; 482-489 (1981))), which arepart of the GCG software packet [Genetics Computer Group, 575 ScienceDrive, Madison, Wis., USA 53711 (1991)], are to be used. The sequenceidentity values recited above in percent (%) are to be determined,preferably, using the program GAP over the entire sequence region withthe following settings: Gap Weight: 50, Length Weight: 3, Average Match:10.000 and Average Mismatch: 0.000, which, unless otherwise specified,shall always be used as standard settings for sequence alignments.

A polynucleotide comprising a fragment of any of the aforementionednucleic acid sequences is also encompassed as a polynucleotide of thepresent invention. The fragment shall encode a polypeptide which stillhas ω-3 desaturase activity as specified above. Accordingly, thepolypeptide may comprise or consist of the domains of the polypeptide ofthe present invention conferring the said biological activity. Afragment as meant herein, preferably, comprises at least 50, at least100, at least 250 or at least 500 consecutive nucleotides of any one ofthe aforementioned nucleic acid sequences or encodes an amino acidsequence comprising at least 20, at least 30, at least 50, at least 80,at least 100 or at least 150 consecutive amino acids of any one of theaforementioned amino acid sequences.

The variant polynucleotides or fragments referred to above, preferably,encode polypeptides retaining at least 10%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80% orat least 90% of the ω-3 desaturase activity exhibitited by thepolypeptide shown in SEQ ID NO: 2 or 24. The activity may be tested asdescribed in the accompanying Examples.

Further varaiant polynucleotides encompassed by the present inventioncomprise sequence motifs as shown in any one of SEQ ID NOs: 15, 16, 17,18, 19, 20, 21, 22, 37, 38, 39, 40, 41, 42, 43, 44 or 45. The depictedsequences show amino acid sequence patterns (also referred to aspolypeptide patterns) which are required for a polynucleotide in orderto encode a polypeptide having ω-3 desaturase activity as specifiedabove and, in particular, for those polypeptides being capable ofconverting ω-6 DPA into DHA. In principle, a polypeptide pattern asreferred to in accordance with the present invention comprises,preferably, less than 100 or less than 50, more preferably, at least 10up to 30 or at least 15 up to 20 amino acid in length. Moreover, it isto be understood that a variant polynucleotide comprised by the presentinvention, preferably, comprises at least one, at least two, at leastthree, at least four, at least five, at least six, at least seven, atleast eight, at least nine, at least ten, at least eleven, at leasttwelve, at least thirteen, at least fourteen, at least fifteen, at leastsixteen or all of the aforementioned specific sequence motifs.Accordingly, the pattern as shown in SEQ ID NO: 15 may be combined withthe pattern shown in SEQ ID NO: 16, the pattern as shown in SEQ ID NO:16 may be combined with the pattern shown in SEQ ID NO: 17, the patternas shown in SEQ ID NO: 17 may be combined with the pattern shown in SEQID NO: 18, the pattern as shown in SEQ ID NO: 18 may be combined withthe pattern shown in SEQ ID NO: 19, the pattern as shown in SEQ ID NO:19 may be combined with the pattern shown in SEQ ID NO: 20. Likewise,the pattern as shown in SEQ ID NO: 37 may be combined with the patternshown in SEQ ID NO: 38, the pattern as shown in SEQ ID NO: 38 may becombined with the pattern shown in SEQ ID NO: 49, the pattern as shownin SEQ ID NO: 22 may be combined with the pattern shown in SEQ ID NO: 37or the pattern as shown in SEQ ID NO: 20 may be combined with thepattern shown in SEQ ID NO: 37 and the pattern as shown in SEQ ID NO:44. In principle, all permutations for the combination of pairs orgroups of up to seventeen patterns based on the aforementioned sequencepattern are envisaged by the present invention.

The polynucleotides of the present invention either essentially consistof the aforementioned nucleic acid sequences or comprise theaforementioned nucleic acid sequences. Thus, they may contain furthernucleic acid sequences as well. Preferably, the polynucleotide of thepresent invention may comprise further untranslated sequence at the 3′and at the 5′ terminus of the coding gene region: at least 500,preferably 200, more preferably 100 nucleotides of the sequence upstreamof the 5′ terminus of the coding region and at least 100, preferably 50,more preferably 20 nucleotides of the sequence downstream of the 3′terminus of the coding gene region. Furthermore, the polynucleotides ofthe present invention may encode fusion proteins wherein one partner ofthe fusion protein is a polypeptide being encoded by a nucleic acidsequence recited above. Such fusion proteins may comprise as additionalpart other enzymes of the fatty acid or lipid biosynthesis pathways,polypeptides for monitoring expression (e.g., green, yellow, blue or redfluorescent proteins, alkaline phosphatase and the like) or so called“tags” which may serve as a detectable marker or as an auxiliary measurefor purification purposes. Tags for the different purposes are wellknown in the art and comprise FLAG-tags, 6-histidine-tags, MYC-tags andthe like.

Variant polynucleotides as referred to in accordance with the presentinvention may be obtained by various natural as well as artificialsources. For example, polynucleotides may be obtained by in vitro and invivo mutagenesis approaches using the above mentioned mentioned specificpolynucleotides as a basis. Moreover, polynucleotids being homologs ororthologs may be obtained from various animal, plant or fungus species.Preferably, they are obtained from plants such as algae, for exampleIsochrysis, Mantoniella, Ostreococcus or Crypthecodinium, algae/diatomssuch as Phaeodactylum or Thraustochytrium, mosses such as Physcomitrellaor Ceratodon, or higher plants such as the Primulaceae such asAleuritia, Calendula stellata, Osteospermum spinescens or Osteospermumhyoseroides, microorganisms such as fungi, such as Aspergillus,Thraustochytrium, Phytophthora, Entomophthora, Mucor or Mortierella,bacteria such as Shewanella, yeasts or animals. Preferred animals arenematodes such as Caenorhabditis, insects or vertebrates. Among thevertebrates, the polynucleotides may, preferably, be derived fromEuteleostomi, Actinopterygii; Neopterygii; Teleostei; Euteleostei,Protacanthopterygii, Salmoniformes; Salmonidae or Oncorhynchus, morepreferably, from the order of the Salmoniformes, most preferably, thefamily of the Salmonidae, such as the genus Salmo, for example from thegenera and species Oncorhynchus mykiss, Trutta trutta or Salmo truttafario. Moreover, the polynucleotides may be obtained from the diatomssuch as the genera Thallasiosira or Crypthecodinium.

The polynucleotide of the present invention shall be provided,preferably, either as an isolated polynucleotide (i.e. isolated from itsnatural context such as a gene locus) or in genetically modified form.An isolated polynucleotide can, for example, comprise less thanapproximately 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb ofnucleotide sequences which naturally flank the nucleic acid molecule inthe genomic DNA of the cell from which the nucleic acid is derived. Thepolynucleotide, preferably, is double or single stranded DNA includingcDNA or RNA. The term encompasses single as well as double strandedpolynucleotides. Moreover, comprised are also chemically modifiedpolynucleotides including naturally occurring modified polynucleotidessuch as glycosylated or methylated polynucleotides or artificialmodified ones such as biotinylated polynucleotides.

Advantageously, it has been found in the studies underlying the presentinvention that the polypeptides being encoded by the polynucleotides ofthe present invention have ω-3 desaturse activity and, in particular,are capable of converting ω-6 LCPUFA substrates, such as C20- andC22-PUFAs, into the corresponding ω-3 PUFAs. As shown in Table 1 in theaccompanying Examples, the conversion of ARA into EPA is catalyzed withthe highest efficiency (more than 40%). However, the conversion of DGLAinto ETA is also catalyzed. Remarkably, the enzymes encoded by thepolynucleotides of the present invention are even capable of catalyzingthe conversion of DPA into DHA. The polynucleotides of the presentinvention are, in principle, useful for the synthesis of LCPUFAs andcompositions containing such compounds. Specifically, thanks to thepresent invention, LCPUFAs and, in particular, even DHA can berecombinantly manufactured using transgenic organisms, such asmicro-organisms, plants and animals.

The present invention also relates to a vector comprising thepolynucleotide of the present invention.

The term “vector”, preferably, encompasses phage, plasmid, viral orretroviral vectors as well as artificial chromosomes, such as bacterialor yeast artificial chromosomes. Moreover, the term also relates totargeting constructs which allow for random or site-directed integrationof the targeting construct into genomic DNA. Such target constructs,preferably, comprise DNA of sufficient length for either homolgous orheterologous recombination as described in detail below. The vectorencompassing the polynucleotides of the present invention, preferably,further comprises selectable markers for propagation and/or selection ina host. The vector may be incorporated into a host cell by varioustechniques well known in the art. If introduced into a host cell, thevector may reside in the cytoplasm or may be incorporated into thegenome. In the latter case, it is to be understood that the vector mayfurther comprise nucleic acid sequences which allow for homologousrecombination or heterologous insertion. Vectors can be introduced intoprokaryotic or eukaryotic cells via conventional transformation ortransfection techniques. The terms “transformation” and “transfection”,conjugation and transduction, as used in the present context, areintended to comprise a multiplicity of prior-art processes forintroducing foreign nucleic acid (for example DNA) into a host cell,including calcium phosphate, rubidium chloride or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection,natural competence, carbon-based clusters, chemically mediated transfer,electroporation or particle bombardment. Suitable methods for thetransformation or transfection of host cells, including plant cells, canbe found in Sambrook et al. (Molecular Cloning: A Laboratory Manual,2^(nd) ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989) and other laboratory manuals,such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacteriumprotocols, Ed.: Gartland and Davey, Humana Press, Totowa, N.J.Alternatively, a plasmid vector may be introduced by heat shock orelectroporation techniques. Should the vector be a virus, it may bepackaged in vitro using an appropriate packaging cell line prior toapplication to host cells. Retroviral vectors may be replicationcompetent or replication defective. In the latter case, viralpropagation generally will occur only in complementing host/cells.

Preferably, the vector referred to herein is suitable as a cloningvector, i.e. replicable in microbial systems. Such vectors ensureefficient cloning in bacteria and, preferably, yeasts or fungi and makepossible the stable transformation of plants. Those which must bementioned are, in particular, various binary and co-integrated vectorsystems which are suitable for the T-DNA-mediated transformation. Suchvector systems are, as a rule, characterized in that they contain atleast the vir genes, which are required for the Agrobacterium-mediatedtransformation, and the sequences which delimit the T-DNA (T-DNAborder). These vector systems, preferably, also comprise furthercis-regulatory regions such as promoters and terminators and/orselection markers with which suitable transformed host cells ororganisms can be identified. While co-integrated vector systems have virgenes and T-DNA sequences arranged on the same vector, binary systemsare based on at least two vectors, one of which bears vir genes, but noT-DNA, while a second one bears T-DNA, but no vir gene. As aconsequence, the last-mentioned vectors are relatively small, easy tomanipulate and can be replicated both in E. coli and in Agrobacterium.These binary vectors include vectors from the pBIB-HYG, pPZP, pBecks,pGreen series. Preferably used in accordance with the invention areBin19, pBI101, pBinAR, pGPTV and pCAMBIA. An overview of binary vectorsand their use can be found in Hellens et al, Trends in Plant Science(2000) 5, 446-451. Furthermore, by using appropriate cloning vectors,the polynucleotides can be introduced into host cells or organisms suchas plants or animals and, thus, be used in the transformation of plants,such as those which are published, and cited, in: Plant MolecularBiology and Biotechnology (CRC Press, Boca Raton, Fla.), chapter 6/7,pp. 71-119 (1993); F. F. White, Vectors for Gene Transfer in HigherPlants; in: Transgenic Plants, vol. 1, Engineering and Utilization, Ed.:Kung and R. Wu, Academic Press, 1993, 15-38; B. Jenes et al., Techniquesfor Gene Transfer, in: Transgenic Plants, vol. 1, Engineering andUtilization, Ed.: Kung and R. Wu, Academic Press (1993), 128-143;Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991),205-225.

More preferably, the vector of the present invention is an expressionvector. In such an expression vector, the polynucleotide is operativelylinked to expression control sequences (also called “expressioncassette”) allowing expression in prokaryotic or eukaryotic cells orisolated fractions thereof. Expression of said polynucleotide comprisestranscription of the polynucleotide, preferably, into a translatablemRNA. Regulatory elements ensuring expression in eukaryotic cells,preferably mammalian cells, are well known in the art. They, preferably,comprise regulatory sequences ensuring initiation of transcription and,optionally, poly-A signals ensuring termination of transcription andstabilization of the transcript. Additional regulatory elements mayinclude transcriptional as well as translational enhancers. Possibleregulatory elements permitting expression in prokaryotic host cellscomprise, e.g., the lac, trp or tac promoter in E. coli, and examplesfor regulatory elements permitting expression in eukaryotic host cellsare the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-, RSV-promoter(Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron inmammalian and other animal cells. Moreover, inducible expression controlsequences may be used in an expression vector encompassed by the presentinvention. Such inducible vectors may comprise tet or lac operatorsequences or sequences inducible by heat shock or other environmentalfactors. Suitable expression control sequences are well known in theart. Beside elements which are responsible for the initiation oftranscription such regulatory elements may also comprise transcriptiontermination signals, such as the SV40-poly-A site or the tk-poly-A site,downstream of the polynucleotide. Preferably, the expression vector isalso a gene transfer or targeting vector. Expression vectors derivedfrom viruses such as retroviruses, vaccinia virus, adeno-associatedvirus, herpes viruses, or bovine papilloma virus, may be used fordelivery of the polynucleotides or vector of the invention into targetedcell population. Methods which are well known to those skilled in theart can be used to construct recombinant viral vectors; see, forexample, the techniques described in Sambrook, Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. andAusubel, Current Protocols in Molecular Biology, Green PublishingAssociates and Wiley Interscience, N.Y. (1994).

Suitable expression vectors are known in the art such as Okayama-BergcDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3(Invitrogene) or pSPORT1 (GIBCO BRL). Further examples of typical fusionexpression vectors are pGEX (Pharmacia Biotech Inc; Smith, D. B., andJohnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.), whereglutathione S-transferase (GST), maltose E-binding protein and proteinA, respectively, are fused with the recombinant target protein. Examplesof suitable inducible nonfusion E. coli expression vectors are, interalia, pTrc (Amann et al. (1988) Gene 69:301-315) and pET 11d (Studier etal., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). The target gene expression ofthe pTrc vector is based on the transcription from a hybrid trp-lacfusion promoter by host RNA polymerase. The target gene expression fromthe pET 11d vector is based on the transcription of a T7-gn10-lac fusionpromoter, which is mediated by a coexpressed viral RNA polymerase (T7gn1). This viral polymerase is provided by the host strains BL21 (DE3)or HMS174 (DE3) from a resident λ-prophage which harbors a T7 gn1 geneunder the transcriptional control of the lacUV 5 promoter. The skilledworker is familiar with other vectors which are suitable in prokaryoticorganisms; these vectors are, for example, in E. coli, pLG338, pACYC184,the pBR series such as pBR322, the pUC series such as pUC18 or pUC19,the M113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200,pUR290, pIN-III113-B1, λgt11 or pBdCl, in Streptomyces plJ101, plJ364,plJ702 or plJ361, in Bacillus pUB110, pC194 or pBD214, inCorynebacterium pSA77 or pAJ667. Examples of vectors for expression inthe yeast S. cerevisiae comprise pYeDesaturasec1 (Baldari et al. (1987)Embo J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943),pJRY88 (Schultz et al. (1987) Gene 54:113-123) and pYES2 (InvitrogenCorporation, San Diego, Calif.). Vectors and processes for theconstruction of vectors which are suitable for use in other fungi, suchas the filamentous fungi, comprise those which are described in detailin: van den Hondel, C. A. M. J. J., & Punt, P. J. (1991) “Gene transfersystems and vector development for filamentous fungi, in: AppliedMolecular Genetics of fungi, J. F. Peberdy et al., Ed., pp. 1-28,Cambridge University Press: Cambridge, or in: More Gene Manipulations inFungi (J. W. Bennett & L. L. Lasure, Ed., pp. 396-428: Academic Press:San Diego). Further suitable yeast vectors are, for example, pAG-1,YEp6, YEp13 or pEMBLYe23. As an alternative, the polynucleotides of thepresent invention can be also expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors which are availablefor the expression of proteins in cultured insect cells (for example Sf9cells) comprise the pAc series (Smith et al. (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39).

The following promoters and expression control sequences may be,preferably, used in an expression vector according to the presentinvention. The cos, tac, trp, tet, trp-tet, Ipp, lac, lpp-lac, laclq,T7, T5, T3, gal, trc, ara, SP6, λ-PR or λ-PL promoters are, preferably,used in Gram-negative bacteria. For Gram-positive bacteria, promotersamy and SPO2 may be used. From yeast or fungal promoters ADC1, MFα, AC,P-60, CYC1, GAPDH, TEF, rp28, ADH are, preferably, used or from plantthe promoters CaMV/35S [Franck et al., Cell 21 (1980) 285-294], PRP1[Ward et al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, lib4, usp, STLS1,B33, nos or the ubiquitin or phaseolin promoter. Also preferred in thiscontext are inducible promoters, such as the promoters described in EP A0 388 186 (benzylsulfonamide-inducible), Plant J. 2, 1992:397-404 (Gatzet al., tetracyclin-inducible), EP A 0 335 528 (abscisic-acid-inducible)or WO 93/21334 (ethanol- or cyclohexenol-inducible). Further suitableplant promoters are the promoter of cytosolic FBPase or the ST-LSIpromoter from potato (Stockhaus et al., EMBO J. 8, 1989, 2445), thephosphoribosyl-pyrophosphate amidotransferase promoter from Glycine max(Genbank accession No. U87999) or the node-specific promoter describedin EP-A-0 249 676. Particularly preferred are promoters which enable theexpression in tissues which are involved in the biosynthesis of fattyacids. Also particularly preferred are seed-specific promoters such asthe USP promoter in accordance with the practice, but also otherpromoters such as the LeB4, DC3, phaseolin or napin promoters. Furtherespecially advantageous promoters are seed-specific promoters which canbe used for monocotyledonous or dicotyledonous plants and which aredescribed in U.S. Pat. No. 5,608,152 (napin promoter from oilseed rape),WO 98/45461 (oleosin promoter from Arobidopsis, U.S. Pat. No. 5,504,200(phaseolin promoter from Phaseolus vulgaris), WO 91/13980 (Bce4 promoterfrom Brassica), by Baeumlein et al., Plant J., 2, 2, 1992:233-239 (LeB4promoter from a legume), these promoters being suitable for dicots. Thefollowing promoters are suitable for example for monocots: lpt-2 orlpt-1 promoter from barley (WO 95/15389 and WO 95/23230), hordeinpromoter from barley and other promoters which are suitable and whichare described in WO 99/16890. In principle, it is possible to use allnatural promoters together with their regulatory sequences, such asthose mentioned above, for the novel process. Likewise, it is possibleand advantageous to use synthetic promoters, either additionally oralone, especially when they mediate a seed-specific expression, such as,for example, as described in WO 99/16890.

The polynucleotides of the present invention can be expressed insingle-cell plant cells (such as algae), see Falciatore et al., 1999,Marine Biotechnology 1 (3):239-251 and the references cited therein, andplant cells from higher plants (for example Spermatophytes, such asarable crops) by using plant expression vectors. Examples of plantexpression vectors comprise those which are described in detail in:Becker, D., Kemper, E., Schell, J., and Masterson, R. (1992) “New plantbinary vectors with selectable markers located proximal to the leftborder”, Plant Mol. Biol. 20:1195-1197; and Bevan, M. W. (1984) “BinaryAgrobacterium vectors for plant transformation”, Nucl. Acids Res.12:8711-8721; Vectors for Gene Transfer in Higher Plants; in: TransgenicPlants, Vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu,Academic Press, 1993, p. 15-38. A plant expression cassette, preferably,comprises regulatory sequences which are capable of controlling the geneexpression in plant cells and which are functionally linked so that eachsequence can fulfill its function, such as transcriptional termination,for example polyadenylation signals. Preferred polyadenylation signalsare those which are derived from Agrobacterium tumefaciens T-DNA, suchas the gene 3 of the Ti plasmid pTiACH5, which is known as octopinesynthase (Gielen et al., EMBO J. 3 (1984) 835 et seq.) or functionalequivalents of these, but all other terminators which are functionallyactive in plants are also suitable. Since plant gene expression is veryoften not limited to transcriptional levels, a plant expression cassettepreferably comprises other functionally linked sequences such astranslation enhancers, for example the overdrive sequence, whichcomprises the 5′-untranslated tobacco mosaic virus leader sequence,which increases the protein/RNA ratio (Gallie et al., 1987, Nucl. AcidsResearch 15:8693-8711). As described above, plant gene expression mustbe functionally linked to a suitable promoter which performs theexpression of the gene in a timely, cell-specific or tissue-specificmanner. Promoters which can be used are constitutive promoters (Benfeyet al., EMBO J. 8 (1989) 2195-2202) such as those which are derived fromplant viruses such as 35S CAMV (Franck et al., Cell 21 (1980) 285-294),19S CaMV (see also U.S. Pat. No. 5,352,605 and WO 84/02913) or plantpromoters such as the promoter of the Rubisco small subunit, which isdescribed in U.S. Pat. No. 4,962,028. Other preferred sequences for theuse in functional linkage in plant gene expression cassettes aretargeting sequences which are required for targeting the gene productinto its relevant cell compartment (for a review, see Kermode, Crit.Rev. Plant Sci. 15, 4 (1996) 285-423 and references cited therein), forexample into the vacuole, the nucleus, all types of plastids, such asamyloplasts, chloroplasts, chromoplasts, the extracellular space, themitochondria, the endoplasmic reticulum, oil bodies, peroxisomes andother compartments of plant cells. As described above, plant geneexpression can also be facilitated via a chemically inducible promoter(for a review, see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol.Biol., 48:89-108). Chemically inducible promoters are particularlysuitable if it is desired that genes are expressed in a time-specificmanner. Examples of such promoters are a salicylic-acid-induciblepromoter (WO 95/19443), a tetracyclin-inducible promoter (Gatz et al.(1992) Plant J. 2, 397-404) and an ethanol-inducible promoter. Promoterswhich respond to biotic or abiotic stress conditions are also suitablepromoters, for example the pathogen-induced PRP1-gene promoter (Ward etal., Plant Mol. Biol. 22 (1993) 361-366), the heat-inducible hsp80promoter from tomato (U.S. Pat. No. 5,187,267), the cold-induciblealpha-amylase promoter from potato (WO 96/12814) or the wound-induciblepinII promoter (EP A 0 375 091). The promoters which are especiallypreferred are those which bring about the expression of genes in tissuesand organs in which fatty acid, lipid and oil biosynthesis takes place,in seed cells such as the cells of endosperm and of the developingembryo. Suitable promoters are the napin gene promoter from oilseed rape(U.S. Pat. No. 5,608,152), the USP promoter from Vicia faba (Baeumleinet al., Mol. Gen. Genet., 1991, 225 (3):459-67), the oleosin promoterfrom Arabidopsis (WO 98/45461), the phaseolin promoter from Phaseolusvulgaris (U.S. Pat. No. 5,504,200), the Bce4 promoter from Brassica (WO91/13980) or the legumin B4 promoter (LeB4; Baeumlein et al., 1992,Plant Journal, 2 (2):233-9), and promoters which bring about theseed-specific expression in monocotyledonous plants such as maize,barley, wheat, rye, rice and the like. Suitable promoters to be takeninto consideration are the lpt2 or lpt1 gene promoter from barley (WO95/15389 and WO 95/23230) or those which are described in WO 99/16890(promoters from the barley hordein gene, the rice glutelin gene, therice oryzin gene, the rice prolamin gene, the wheat gliadin gene, wheatglutelin gene, the maize zein gene, the oat glutelin gene, the sorghumkasirin gene, the rye secalin gene). Likewise, especially suitable arepromoters which bring about the plastid-specific expression sinceplastids are the compartment in which the precursors and some endproducts of lipid biosynthesis are synthesized. Suitable promoters suchas the viral RNA-polymerase promoter, are described in WO 95/16783 andWO 97/06250, and the clpP promoter from Arabidopsis, described in WO99/46394.

The abovementioned vectors are only a small overview of vectors to beused in accordance with the present invention. Further vectors are knownto the skilled worker and are described, for example, in: CloningVectors (Ed., Pouwels, P. H., et al., Elsevier, Amsterdam-NewYork-Oxford, 1985, ISBN 0 444 904018). For further suitable expressionsystems for prokaryotic and eukaryotic cells see the chapters 16 and 17of Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: ALaboratory Manual, 2^(nd) edition, Cold Spring Harbor Laboratory, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In a preferred embodiment of the vector of the present invention, thesaid vector comprises at least one polynucleotide encoding a furtherenzyme being involved in the bio-synthesis of fatty acids or lipids. Afurther enzyme referred to in accordance with the present invention is,preferably, selected from the group consisting of acyl-CoAdehydrogenase(s), acyl-ACP [=acyl carrier protein] desaturase(s),acyl-ACP thio-esterase(s), fatty acid acyltransferase(s),acyl-CoA:lysophospholipid acyltransferase(s), fatty acid synthase(s),fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s),acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acidacetylenase(s), lipoxy-genase(s), triacylglycerol lipase(s), allenoxidesynthase(s), hydroperoxide lyase(s) or fatty acid elongase(s),acyl-CoA:lysophospholipid acyltransferase, Δ4-desaturase, Δ5-desaturase,Δ6-desaturase, Δ8-desaturase, Δ9-desaturase, Δ12-desaturase,Δ5-elongase, Δ6-elongase and Δ9-elongase. Most preferably, the vectorcomprises at least one polynucleotide encoding an enzyme selected fromthe group consisting of Δ4-desaturase, Δ5-desaturase, Δ6-desaturase,Δ8-desaturase, Δ9-desaturase, Δ12-desaturase Δ5-elongase, Δ6-elongaseand Δ9-elongase in addition to at least one polynucleotide encoding anenzyme selected from the group consisting of acyl-CoA dehydrogenase(s),acyl-ACP [=acyl carrier protein] desaturase(s), acyl-ACPthio-esterase(s), fatty acid acyltransferase(s),acyl-CoA:lysophospholipid acyltransferase(s), fatty acid synthase(s),fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s),acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acidacetylenase(s), lipoxy-genase(s), triacylglycerol lipase(s), allenoxidesynthase(s), hydroperoxide lyase(s) or fatty acid elongase(s), andacyl-CoA:lysophospholipid acyltransferase. The at least onepolynucleotide encoding said further enzyme may be obtained from anybacteria, fungi, animal or plant and, preferably, from thosespecifically recited in this description. Preferably, eachpolynucleotide encoding a further enzyme as recited above is also linkedto its own expression control sequence wherein said expression controlsequences may or may not be identical. The vector of the presentinvention, thus, preferably, comprises at least two (i.e. the expressioncassette for the polynucleotide of the present invention and thepolynucleotide for the at least one further enzyme) up to a plurality ofexpression cassettes consisting of the polynucleotides and expressioncontrol sequences operatively linked expression control sequencesthereto.

The invention also pertains to a host cell comprising the polynucleotideor the vector of the present invention.

Host cells are primary cells or cell lines derived from multicellularorganisms such as plants or animals. Furthermore, host cells encompassprokaryotic or eukaryotic single cell organisms (also referred to asmicro-organisms). Primary cells or cell lines to be used as host cellsin accordance with the present invention may be derived from themulticellular organisms referred to below. Host cells which can beexploited are furthermore mentioned in: Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). Specific expression strains which can be used, for example thosewith a lower protease activity, are described in: Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128. These include plant cells and certaintissues, organs and parts of plants in all their phenotypic forms suchas anthers, fibers, root hairs, stalks, embryos, calli, cotelydons,petioles, harvested material, plant tissue, reproductive tissue and cellcultures which is derived from the actual transgenic plant and/or can beused for bringing about the transgenic plant. Preferably, the host cellsmay be obtained from plants. More preferably, oil crops are envisagedwhich comprise large amounts of lipid compounds, such as oilseed rape,evening primrose, hemp, thistle, peanut, canola, linseed, soybean,safflower, sunflower, borage, or plants such as maize, wheat, rye, oats,triticale, rice, barley, cotton, cassava, pepper, Tagetes, Solanaceaeplants such as potato, tobacco, eggplant and tomato, Vicia species, pea,alfalfa, bushy plants (coffee, cacao, tea), Salix species, trees (oilpalm, coconut) and perennial grasses and fodder crops. Especiallypreferred plants according to the invention are oil crops such assoybean, peanut, oilseed rape, canola, linseed, hemp, evening primrose,sunflower, safflower, trees (oil palm, coconut). Suitable methods forobtaining host cells from the multicellular organisms referred to belowas well as conditions for culturing these cells are well known in theart.

The micro-organisms are, preferably, bacteria or fungi including yeasts.Preferred fungi to be used in accordance with the present invention areselected from the group of the families Chaetomiaceae, Choanephoraceae,Cryptococcaceae, Cunninghamellaceae, Demetiaceae, Moniliaceae,Mortierellaceae, Mucoraceae, Pythiaceae, Sacharomycetaceae,Saprolegniaceae, Schizosacharomycetaceae, Sodariaceae orTuberculariaceae. Further preferred micro-organisms are selected fromthe group: Choanephoraceae such as the genera Blakeslea, Choanephora,for example the genera and species Blakeslea trispora, Choanephoracucurbitarum, Choanephora infundibulifera var. cucurbitarum,Mortierellaceae, such as the genus Mortierella, for example the generaand species Mortierella isabeffina, Mortierella polycephala, Mortierellaramanniana, Mortierella vinacea, Mortierella zonata, Pythiaceae such asthe genera Phytium, Phytophthora for example the genera and speciesPythium debaryanum, Pythium intermedium, Pythium irregulare, Pythiummegalacanthum, Pythium paroecandrum, Pythium sylvaticum, Pythiumultimum, Phytophthora cactorum, Phytophthora cinnamomi, Phytophthoracitricola, Phytophthora citrophthora, Phytophthora cryptogea,Phytophthora drechsleri, Phytophthora erythroseptica, Phytophthoralateralis, Phytophthora megasperma, Phytophthora nicotianae,Phytophthora nicotianae var. parasitica, Phytophthora palmivora,Phytophthora parasitica, Phytophthora syringae, Saccharomycetaceae suchas the genera Hansenula, Pichia, Saccharomyces, Saccharomycodes,Yarrowia for example the genera and species Hansenula anomala, Hansenulacafifomica, Hansenula canadensis, Hansenula capsulata, Hansenulaciferrii, Hansenula glucozyma, Hansenula henricii, Hansenula holstfi,Hansenula minuta, Hansenula nonfermentans, Hansenula philodendri,Hansenula polymorpha, Hansenula saturnus, Hansenula subpefficulosa,Hansenula wickerhamii, Hansenula wingei, Pichia alcoholophila, Pichiaangusta, Pichia anomala, Pichia bispora, Pichia burtonfi, Pichiacanadensis, Pichia capsulata, Pichia carsonii, Pichia cellobiosa, Pichiaciferrii, Pichia farinosa, Pichia fermentans, Pichia finlandica, Pichiaglucozyma, Pichia guiffiermondii, Pichia haplophila, Pichia henricii,Pichia holstfi, Pichia jadinii, Pichia findnerfi, Pichiamembranaefaciens, Pichia methanolica, Pichia minuta var. minuta, Pichiaminuta var. nonfermentans, Pichia norvegensis, Pichia ohmeri, Pichiapastoris, Pichia philodendri, Pichia pini, Pichia polymorpha, Pichiaquercuum, Pichia rhodanensis, Pichia sargentensis, Pichia stipitis,Pichia strasburgensis, Pichia subpefficulosa, Pichia toletana, Pichiatrehalophila, Pichia vini, Pichia xylosa, Saccharomyces aceta,Saccharomyces ball, Saccharomyces bayanus, Saccharomyces bisporus,Saccharomyces capensis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces cerevisiae var. ellipsoideus, Saccharomyceschevalieri, Saccharomyces delbrueckii, Saccharomyces diastaticus,Saccharomyces drosophilarum, Saccharomyces elegans, Saccharomycesellipsoideus, Saccharomyces fermentati, Saccharomyces florentinus,Saccharomyces fragilis, Saccharomyces heterogenicus, Saccharomyceshienipiensis, Saccharomyces inusitatus, Saccharomyces italicus,Saccharomyces kluyveri, Saccharomyces krusei, Saccharomyces lactis,Saccharomyces marxianus, Saccharomyces microellipsoides, Saccharomycesmontanus, Saccharomyces norbensis, Saccharomyces oleaceus, Saccharomycesparadoxus, Saccharomyces pastorianus, Saccharomyces pretoriensis,Saccharomyces rosei, Saccharomyces rouxii, Saccharomyces uvarum,Saccharomycodes ludwigii, Yarrowia lipolytica, Schizosacharomycetaceaesuch as the genera Schizosaccharomyces e.g. the speciesSchizosaccharomyces japonicus var. japonicus, Schizosaccharomycesjaponicus var. versatilis, Schizosaccharomyces malidevorans,Schizosaccharomyces octosporus, Schizosaccharomyces pombe var.malidevorans, Schizosaccharomyces pombe var. pombe, Thraustochytriaceaesuch as the genera Althornia, Aplanochytrium, Japonochytrium,Schizochytriurn, Thraustochytrium e.g. the species Schizochytriumaggregatum, Schizochytrium limacinum, Schizochytrium mangrovei,Schizochytrium minutum, Schizochytrium octosporum, Thraustochytriumaggregatum, Thraustochytrium amoeboideum, Thraustochytrium antacticum,Thraustochytrium arudimentale, Thraustochytrium aureum, Thraustochytriumbenthicola, Thraustochytrium globosum, Thraustochytrium indicum,Thraustochytrium kerguelense, Thraustochytrium kinnei, Thraustochytriummotivum, Thraustochytrium multirudimentale, Thraustochytriumpachydermum, Thraustochytrium proliferum, Thraustochytrium roseum,Thraustochytrium rossii, Thraustochytrium striatum or Thraustochytriumvisurgense. Further preferred microorganisms are bacteria selected fromthe group of the families Bacillaceae, Enterobacteriacae orRhizobiaceae. Examples of such micro-organisms may be selected from thegroup: Bacillaceae such as the genera Bacillus for example the generaand species Bacillus acidocaldarius, Bacillus acidoterrestris, Bacillusalcalophilus, Bacillus amyloliquefaciens, Bacillus amylolyticus,Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacilluscoagulans, Bacillus sphaericus subsp. fusiformis, Bacillusgalactophilus, Bacillus globisporus, Bacillus globisporus subsp.marinus, Bacillus halophilus, Bacillus lentimorbus, Bacillus lentus,Bacillus licheniformis, Bacillus megaterium, Bacillus polymyxa, Bacilluspsychrosaccharolyticus, Bacillus pumilus, Bacillus sphaericus, Bacillussubtilis subsp. spizizenii, Bacillus subtilis subsp. subtilis orBacillus thuringiensis; Enterobacteriacae such as the generaCitrobacter, Edwardsiella, Enterobacter, Erwinia, Escherichia,Klebsiella, Salmonella or Serratia for example the genera and speciesCitrobacter amalonaticus, Citrobacter diversus, Citrobacter freundii,Citrobacter genomospecies, Citrobacter gillenii, Citrobacterintermedium, Citrobacter koseri, Citrobacter murliniae, Citrobacter sp.,Edwardsiella hoshinae, Edwardsiella ictaluri, Edwardsiella tarda,Erwinia alni, Erwinia amylovora, Erwinia ananatis, Erwinia aphidicola,Erwinia billingiae, Erwinia cacticida, Erwinia cancerogena, Erwiniacarnegieana, Erwinia carotovora subsp. atroseptica, Erwinia carotovorasubsp. betavasculorum, Erwinia carotovora subsp. odorifera, Erwiniacarotovora subsp. wasabiae, Erwinia chrysanthemi, Erwinia cypripedii,Erwinia dissolvens, Erwinia herbicola, Erwinia mallotivora, Erwiniamilletiae, Erwinia nigrifluens, Erwinia nimipressuralis, Erwiniapersicina, Erwinia psidii, Erwinia pyrifoliae, Erwinia quercina, Erwiniarhapontici, Erwinia rubrifaciens, Erwinia salicis, Erwinia stewartii,Erwinia tracheiphila, Erwinia uredovora, Escherichia adecarboxylata,Escherichia anindolica, Escherichia aurescens, Escherichia blattae,Escherichia coli, Escherichia coli var. communion, Escherichiacoli-mutabile, Escherichia fergusonii, Escherichia hermannii,Escherichia sp., Escherichia vulneris, Klebsiella aerogenes, Klebsiellaedwardsii subsp. atlantae, Klebsiella omithinolytica, Klebsiellaoxytoca, Klebsiella planticola, Klebsiella pneumoniae, Klebsiellapneumoniae subsp. pneumoniae, Klebsiella sp., Klebsiella terrigena,Klebsiella trevisanii, Salmonella abony, Salmonella arizonae, Salmonellabongori, Salmonella choleraesuis subsp. arizonae, Salmonellacholeraesuis subsp. bongori, Salmonella choleraesuis subsp.cholereasuis, Salmonella choleraesuis subsp. diarizonae, Salmonellacholeraesuis subsp. houtenae, Salmonella choleraesuis subsp. indica,Salmonella choleraesuis subsp. salamae, Salmonella daressalaam,Salmonella enterica subsp. houtenae, Salmonella enterica subsp. salamae,Salmonella enteritidis, Salmonella gallinarum, Salmonella heidelberg,Salmonella panama, Salmonella senftenberg, Salmonella typhimurium,Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratiagrimesii, Serratia liquefaciens, Serratia marcescens, Serratiamarcescens subsp. marcescens, Serratia marinorubra, Serratia odorifera,Serratia plymouthensis, Serratia plymuthica, Serratia proteamaculans,Serratia proteamaculans subsp. quinovora, Serratia quinivorans orSerratia rubidaea; Rhizobiaceae such as the genera Agrobacterium,Carbophilus, Chelatobacter, Ensifer, Rhizobium, Sinorhizobium forexample the genera and species Agrobacterium atlanticum, Agrobacteriumferrugineum, Agrobacterium gelatinovorum, Agrobacterium lanymoorei,Agrobacterium meteori, Agrobacterium radiobacter, Agrobacteriumrhizogenes, Agrobacterium rubi, Agrobacterium stellulatum, Agrobacteriumtumefaciens, Agrobacterium vitis, Carbophilus carboxidus, Chelatobacterheintzii, Ensifer adhaerens, Ensifer arboris, Ensifer fredii, Ensiferkostiensis, Ensifer kummerowiae, Ensifer medicae, Ensifer meliloti,Ensifer saheli, Ensifer terangae, Ensifer xinjiangensis, Rhizobiumciceri Rhizobium etli, Rhizobium fredii, Rhizobium galegae, Rhizobiumgallicum, Rhizobium giardinii, Rhizobium hainanense, Rhizobium huakuii,Rhizobium huautlense, Rhizobium indigoferae, Rhizobium japonicum,Rhizobium leguminosarum, Rhizobium loessense, Rhizobium loti, Rhizobiumlupini, Rhizobium mediterraneum, Rhizobium meliloti, Rhizobiummongolense, Rhizobium phaseoli, Rhizobium radiobacter, Rhizobiumrhizogenes, Rhizobium rubi, Rhizobium sullae, Rhizobium tianshanense,Rhizobium trifolii, Rhizobium tropici, Rhizobium undicola, Rhizobiumvitis, Sinorhizobium adhaerens, Sinorhizobium arboris, Sinorhizobiumfredii, Sinorhizobium kostiense, Sinorhizobium kummerowiae,Sinorhizobium medicae, Sinorhizobium meliloti, Sinorhizobium morelense,Sinorhizobium saheli or Sinorhizobium xinjiangense.

How to culture the aforementioned micro-organisms is well known to theperson skilled in the art.

In a preferred embodiment of the host cell of the present invention, thesaid host cell additionally comprises at least one further enzyme beinginvolved in the biosynthesis of fatty acids or lipids, preferably,selected from the group consisting of: acyl-CoA dehydrogenase(s),acyl-ACP [=acyl carrier protein] desaturase(s), acyl-ACPthioesterase(s), fatty acid acyltransferase(s),acyl-CoA:lysophospholipid acyltransferase(s), fatty acid synthase(s),fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s),acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acidacetylenase(s), lipoxygenase(s), triacylglycerol lipase(s), allenoxidesynthase(s), hydroperoxide lyase(s) or fatty acid elongase(s),acyl-CoA:lysophospholipid acyltransferase, LA-desaturase, Δ5-desaturase,Δ6-desaturase, Δ3-desaturase, Δ9-desaturase, Δ12-desaturase,Δ5-elongase, Δ6-elongase and Δ9-elongase. More preferably, the host cellcomprises at least one further enzyme selected from the group consistingof LA-desaturase, Δ5-desaturase, Δ6-desaturase, Δ3-desaturase,Δ9-desaturase, Δ12-desaturase, Δ5-elongase, Δ6-elongase and Δ9-elongasein addition to at least one further enzyme selected from the groupconsisting of acyl-CoA dehydrogenase(s), acyl-ACP [=acyl carrierprotein] desaturase(s), acyl-ACP thioesterase(s), fatty acidacyltransferase(s), acyl-CoA:lysophospholipid acyltransferase(s), fattyacid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme Acarboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s),fatty acid acetylenase(s), lipoxygenase(s), triacylglycerol lipase(s),allenoxide synthase(s), hydroperoxide lyase(s) or fatty acidelongase(s), and acylCoA:lysophospholipid acyltransferase. The enzymemay be endogenously expressed in the host cell or may be exogenouslysupplied, e.g., by introducing one or more expression vector(s)comprising the polynucleotides encoding the aforementioned furtherenzymes.

The present invention also includes a method for the manufacture of apolypeptide having ω-3 desaturase activity comprising:

-   -   (a) expressing the polynucleotide of the present invention in a        host cell as specified above; and    -   (b) obtaining the polypeptide encoded by said polynucleotide        from the host cell.

The polypeptide may be obtained, for example, by all conventionalpurification techniques including affinity chromatography, sizeexclusion chromatography, high pressure liquid chromatography (HPLC) andprecipitation techniques including antibody precipitation. It is to beunderstood that the method may—although preferred—not necessarily yieldan essentially pure preparation of the polypeptide.

The present invention further relates to a polypeptide encoded by thepolynucleotide of the present invention or which is obtainable by theaforementioned method of the present invention.

The term “polypeptide” as used herein encompasses essentially purifiedpolypeptides or polypeptide preparations comprising other proteins inaddition. Further, the term also relates to the fusion proteins orpolypeptide fragments being at least partially encoded by thepolynucleotide of the present invention referred to above. Moreover, itincludes chemically modified polypeptides. Such modifications may beartificial modifications or naturally occurring modifications such asphosphorylation, glycosylation, myristylation and the like. The terms“polypeptide”, “peptide” or “protein” are used interchangeablethroughout this specification. As referred to above, the polypeptide ofthe present invention shall exhibit ω-3 desaturase activity and, thus,can be used for the manufacture of LCPUFAs, in particular C20- orC22-LCPUFAS, either in a host cell or in a transgenic animal or plant asdescribed elsewhere in this specification. Surprisingly, the ω-3desaturase activity of the polypeptide of the present invention evenincludes the ability to convert ω-6 DPA into DHA.

The present invention also relates to an antibody which specificallyrecognizes the polypeptide of the present invention.

Antibodies against the polypeptides of the invention can be prepared bywell known methods using a purified polypeptide according to theinvention or a suitable fragment derived therefrom as an antigen. Afragment which is suitable as an antigen may be identified byantigenicity determining algorithms well known in the art. Suchfragments may be obtained either from the polypeptide of the inventionby proteolytic digestion or may be a synthetic peptide. Preferably, theantibody of the present invention is a monoclonal antibody, a polyclonalantibody, a single chain antibody, a human or humanized antibody orprimatized, chimerized or fragment thereof. Also comprised as antibodiesby the present invention are a bispecific antibody, a syntheticantibody, an antibody fragment, such as Fab, Fv or scFv fragments etc.,or a chemically modified derivative of any of these. The antibody of thepresent invention shall specifically bind (i.e. does not cross reactwith other polypeptides or peptides) to the polypeptide of theinvention. Specific binding can be tested by various well knowntechniques.

Antibodies or fragments thereof can be obtained by using methods whichare described, e.g., in Harlow and Lane “Antibodies, A LaboratoryManual”, CSH Press, Cold Spring Harbor, 1988. Monoclonal antibodies canbe prepared by the techniques originally described in Köhler andMilstein, Nature 256 (1975), 495, and Galfré, Meth. Enzymol. 73 (1981),3, which comprise the fusion of mouse myeloma cells to spleen cellsderived from immunized mammals.

The antibodies can be used, for example, for the immunoprecipitation,immunolocalization or purification (e.g., by affinity chromatography) ofthe polypeptides of the invention as well as for the monitoring of thepresence of said variant polypeptides, for example, in recombinantorganisms, and for the identification of compounds interacting with theproteins according to the invention.

The present invention relates to a transgenic non-human organismcomprising the polynucleotide, the vector or the host cell of thepresent invention.

The term “non-human transgenic organism”, preferably, relates to aplant, an animal or a multicellular micro-organism. The polynucleotideor vector may be present in the cytoplasm of the organism or may beincorporated into the genome either heterologous or by homologousrecombination. Host cells, in particular those obtained from plants oranimals, may be introduced into a developing embryo in order to obtainmosaic or chimeric organisms, i.e. non-human transgenic organismscomprising the host cells of the present invention. Preferably, thenon-human transgenic organism expresses the polynucleotide of thepresent invention in order to produce the polypeptide in an amountresulting in a detectable ω-3 desaturase activity. Suitable transgenicorganisms are, preferably, all those organisms which are capable ofsynthesizing fatty acids, specifically unsaturated fatty acids, or whichare suitable for the expression of recombinant genes.

Preferred animals to be used for making non-human transgenic organismsaccording to the present invention include mammals, reptiles, birds,fishes, insects and worms. Preferred mammals are rodents such as mice,rabbits or rats or farming animals such as cows, pigs, sheep or goats.Preferred fishes are derived from the classes of the Euteleostomi,Actinopterygii; Neopterygii; Teleostei; Euteleostei,Protacanthopterygii, Salmoniformes; Salmonidae or Oncorhynchus and, morepreferably, from the order of the Salmoniformes, in particular, thefamily of the Salmonidae, such as the genus Salmo, for example from thegenera and species Oncorhynchus mykiss, Trutta trutta or Salmo truttafario. Preferred insects are flies such as the fruitfly Drosophilamelanogaster and preferred worms may be from the family ofCaenorhabditae.

A method for the production of a transgenic non-human animal comprisesintroduction of the polynucleotide or vector of the present inventioninto a germ cell, an embryonic cell, embryonic stem (ES) cell or an eggor a cell derived therefrom. Production of transgenic embryos andscreening of those can be performed, e.g., as described by A. L. JoynerEd., Gene Targeting, A Practical Approach (1993), Oxford UniversityPress. Genomic DNA of embryonic tissues may be analyzed for the presenceof the polynucleotide or vector of the present invention byhybridization-based or PCR-based techniques. A general method for makingtransgenic non-human animals is described in the art, see for example WO94/24274. For making transgenic non-human organisms (which includehomologously targeted non-human animals), ES cells are preferred.Details on making such transgenic non-human organisms are described inRobertson, E. J. (1987) in Teratocarcinomas and Embryonic Stem Cells: APractical Approach. E. J. Robertson, ed. (Oxford: IRL Press), p. 71-112.Methods for producing transgenic insects, such as Drosophilamelanogaster, are also known in the art, see for example U.S. Pat. No.4,670,388, Brand & Perrimon, Development (1993) 118: 401-415; and Phelps& Brand, Methods (April 1998) 14: 367-379. Transgenic nematodes such asC. elegans can be generated as described in Mello, 1991, Embo J 10,3959-70 or Plasterk, 1995 Methods Cell Biol 48, 59-80.

Preferred plants to be used for making non-human transgenic organismsaccording to the present invention are plants which are capable ofsynthesizing fatty acids, such as all dicotyledonous or monocotyledonousplants, algae or mosses. Advantageous plants are selected from the groupof the plant families Adelotheciaceae, Anacardiaceae, Asteraceae,Apiaceae, Betulaceae, Boraginaceae, Brassicaceae, Bromeliaceae,Caricaceae, Cannabaceae, Convolvulaceae, Chenopodiaceae,Crypthecodiniaceae, Cucurbitaceae, Ditrichaceae, Elaeagnaceae,Ericaceae, Euphorbiaceae, Fabaceae, Geraniaceae, Gramineae,Juglandaceae, Lauraceae, Leguminosae, Linaceae, Prasinophyceae orvegetable plants or ornamentals such as Tagetes. Examples which may bementioned are the following plants selected from the group consistingof: Adelotheciaceae such as the genera Physcomitrella, such as the genusand species Physcomitrella patens, Anacardiaceae such as the generaPistacia, Mangifera, Anacardium, for example the genus and speciesPistacia vera [pistachio], Mangifer indica [mango] or Anacardiumoccidentale [cashew], Asteraceae, such as the genera Calendula,Carthamus, Centaurea, Cichorium, Cynara, Helianthus, Lactuca, Locusta,Tagetes, Valeriana, for example the genus and species Calendulaofficinais [common marigold], Carthamus tinctorius [safflower],Centaurea cyanus [cornflower], Cichorium intybus [chicory], Cynarascoiymes [artichoke], Helianthus annus [sunflower], Lactuca sativa,Lactuca crispa, Lactuca esculenta, Lactuca scariola L. ssp. sativa,Lactuca scariola L. var. integrata, Lactuca scariola L. var.integrifolia, Lactuca sativa subsp. romana, Locusta communis, Valerianalocusta [salad vegetables], Tagetes lucida, Tagetes erecta or Tagetestenuifolia [african or french marigold], Apiaceae, such as the genusDaucus, for example the genus and species Daucus carota [carrot],Betulaceae, such as the genus Corylus, for example the genera andspecies Corylus avellana or Corylus columa [hazelnut], Boraginaceae,such as the genus Borago, for example the genus and species Boragoofficinalis [borage], Brassicaceae, such as the genera Brassica,Melanosinapis, Sinapis, Arabadopsis, for example the genera and speciesBrassica napus, Brassica rapa ssp. [oilseed rape], Sinapis arvensisBrassica juncea, Brassica juncea var. juncea, Brassica juncea var.crispifolia, Brassica juncea var. foliosa, Brassica nigra, Brassicasinapioides, Melanosinapis communis [mustard], Brassica oleracea [fodderbeet] or Arabidopsis thaliana, Bromeliaceae, such as the genera Anana,Bromelia (pineapple), for example the genera and species Anana comosus,Ananas ananas or Bromelia comosa [pineapple], Caricaceae, such as thegenus Carica, such as the genus and species Carica papaya [pawpaw],Cannabaceae, such as the genus Cannabis, such as the genus and speciesCannabis sativa [hemp], Convolvulaceae, such as the genera Ipomea,Convolvulus, for example the genera and species Ipomoea batatus, Ipomoeapandurata, Convolvulus batatas, Convolvulus tiliaceus, Ipomoeafastigiata, Ipomoea tiliacea, Ipomoea triloba or Convolvulus panduratus[sweet potato, batate], Chenopodiaceae, such as the genus Beta, such asthe genera and species Beta vulgaris, Beta vulgaris var. altissima, Betavulgaris var. Vulgaris, Beta maritima, Beta vulgaris var. perennis, Betavulgaris var. conditiva or Beta vulgaris var. esculenta [sugarbeet],Crypthecodiniaceae, such as the genus Crypthecodinium, for example thegenus and species Cryptecodinium cohnii, Cucurbitaceae, such as thegenus Cucurbita, for example the genera and species Cucurbita maxima,Cucurbita mixta, Cucurbita pepo or Cucurbita moschata [pumpkin/squash],Cymbellaceae such as the genera Amphora, Cymbella, Okedenia,Phaeodactylum, Reimeria, for example the genus and species Phaeodactylumtricornutum, Ditrichaceae such as the genera Ditrichaceae, Astomiopsis,Ceratodon, Chrysoblastella, Ditrichum, Distichium, Eccremidium,Lophidion, Philibertiella, Pleuridium, Saelania, Trichodon,Skottsbergia, for example the genera and species Ceratodon antarcticus,Ceratodon columbiae, Ceratodon heterophyllus, Ceratodon purpureus,Ceratodon purpureus, Ceratodon purpureus ssp. convolutus, Ceratodon,purpureus spp. stenocarpus, Ceratodon purpureus var. rotundifolius,Ceratodon ratodon, Ceratodon stenocarpus, Chrysoblastella chilensis,Ditrichum ambiguum, Ditrichum brevisetum, Ditrichum crispatissimum,Ditrichum difficile, Ditrichum falcifolium, Ditrichum flexicaule,Ditrichum giganteum, Ditrichum heteromallum, Ditrichum lineare,Ditrichum lineare, Ditrichum montanum, Ditrichum montanum, Ditrichumpallidum, Ditrichum punctulatum, Ditrichum pusillum, Ditrichum pusillumvar. tortile, Ditrichum rhynchostegium, Ditrichum schimperi, Ditrichumtortile, Distichium capillaceum, Distichium hagenii, Distichiuminclinatum, Distichium macounii, Eccremidium floridanum, Eccremidiumwhiteleggei, Lophidion strictus, Pleuridium acuminatum, Pleuridiumalternifolium, Pleuridium holdridgei, Pleuridium mexicanum, Pleuridiumravenelii, Pleuridium subulatum, Saelania glaucescens, Trichodonborealis, Trichodon cylindricus or Trichodon cylindricus var. oblongus,Elaeagnaceae such as the genus Elaeagnus, for example the genus andspecies Olea europaea [olive], Ericaceae such as the genus Kalmia, forexample the genera and species Kalmia latifolia, Kalmia angustifolia,Kalmia microphylla, Kalmia polifolia, Kalmia occidentalis, Cistuschamaerhodendros or Kalmia lucida [mountain laurel], Euphorbiaceae suchas the genera Manihot, Janipha, Jatropha, Ricinus, for example thegenera and species Manihot utilissima, Janipha manihot, Jatrophamanihot, Manihot aipil, Manihot dulcis, Manihot manihot, Manihotmelanobasis, Manihot esculenta [manihot] or Ricinus communis [castor-oilplant], Fabaceae such as the genera Pisum, Albizia, Cathormion,Feuillea, Inga, Pithecolobium, Acacia, Mimosa, Medicajo, Glycine,Dolichos, Phaseolus, Soja, for example the genera and species Pisumsativum, Pisum arvense, Pisum humile [pea], Albizia berteriana, Albiziajulibrissin, Albizia lebbeck, Acacia berteriana, Acacia littoralis,Albizia berteriana, Albizzia berteriana, Cathormion berteriana, Feuilleaberteriana, Inga fragrans, Pithecellobium berterianum, Pithecellobiumfragrans, Pithecolobium berterianum, Pseudalbizzia berteriana, Acaciajulibrissin, Acacia nemu, Albizia nemu, Feuilleea julibrissin, Mimosajulibrissin, Mimosa speciosa, Sericanrda julibrissin, Acacia lebbeck,Acacia macrophylla, Albizia lebbek, Feuilleea lebbeck, Mimosa lebbeck,Mimosa speciosa [silk tree], Medicago sativa, Medicago falcata, Medicagovaria [alfalfa], Glycine max Dolichos soja, Glycine gracilis, Glycinehispida, Phaseolus max, Soja hispida or Soja max [soybean], Funariaceaesuch as the genera Aphanorrhegma, Entosthodon, Funaria, Physcomitrella,Physcomitrium, for example the genera and species Aphanorrhegmaserratum, Entosthodon attenuatus, Entosthodon bolanderi, Entosthodonbonplandii, Entosthodon californicus, Entosthodon drummondii,Entosthodon jamesonii, Entosthodon leibergii, Entosthodon neoscoticus,Entosthodon rubrisetus, Entosthodon spathulifolius, Entosthodon tucsoni,Funaria amedcana, Funaria bolanderi, Funaria calcarea, Funariacalifornica, Funaria calvescens, Funaria convoluta, Funaria flavicans,Funaria groutiana, Funaria hygrometrica, Funaria hygrometrica var.arctica, Funaria hygrometrica var. calvescens, Funaria hygrometrica var.convoluta, Funaria hygrometrica var. muralis, Funaria hygrometrica var.utahensis, Funaria microstoma, Funaria microstoma var. obtusifolia,Funaria muhlenbergii, Funaria orcuttii, Funaria plano-convexa, Funariapolaris, Funaria ravenelii, Funaria rubriseta, Funaria serrata, Funariasonorae, Funaria sublimbatus, Funaria tucsoni, Physcomitrellacalifornica, Physcomitrella patens, Physcomitrella readeri,Physcomitrium australe, Physcomitrium californicum, Physcomitriumcollenchymatum, Physcomitrium coloradense, Physcomitrium cupuliferum,Physcomitrium drummondii, Physcomitrium eurystomum, Physcomitriumflexifolium, Physcomitrium hookeri, Physcomitrium hookeri var. serratum,Physcomitrium immersum, Physcomitrium kellermanii, Physcomitriummegalocarpum, Physcomitrium pyriforme, Physcomitrium pyriforme var.serratum, Physcomitrium rufipes, Physcomitrium sandbergii, Physcomitriumsubsphaericum, Physcomitrium washingtoniense, Geraniaceae, such as thegenera Pelargonium, Cocos, Oleum, for example the genera and speciesCocos nucifera, Pelargonium grossularioides or Oleum cocois [coconut],Gramineae, such as the genus Saccharum, for example the genus andspecies Saccharum officinarum, Juglandaceae, such as the genera Juglans,Wallia, for example the genera and species Juglans regia, Juglansailanthifolia, Juglans sieboldiana, Juglans cinerea, Wallia cinerea,Juglans bixbyi, Jugtans californica, Juglans hindsii, Juglansintermedia, Juglans jamaicensis, Juglans major, Juglans microcarpa,Juglans nigra or Wallia nigra [walnut], Lauraceae, such as the generaPersea, Laurus, for example the genera and species Laurus nobilis [bay],Persea americana, Persea gratissima or Persea persea [avocado],Leguminosae, such as the genus Arachis, for example the genus andspecies Arachis hypogaea [peanut], Linaceae, such as the genera Linum,Adenolinum, for example the genera and species Linum usitatissimum,Linum humile, Linum austriacum, Linum bienne, Linum angustifolium, Linumcatharticum, Linum flavum, Linum grandiflorum, Adenolinum grandiflorum,Linum lewisii, Linum narbonense, Linum perenne, Linum perenne var.lewisii, Linum pratense or Linum trigynum [linseed], Lythrarieae, suchas the genus Punica, for example the genus and species Punica granatum[pomegranate], Malvaceae, such as the genus Gossypium, for example thegenera and species Gossypium hirsutum, Gossypium arboreum, Gossypiumbarbadense, Gossypium herbaceum or Gossypium thurberi [cotton],Marchantiaceae, such as the genus Marchantia, for example the genera andspecies Marchantia berteroana, Marchantia foliacea, Marchantiamacropora, Musaceae, such as the genus Musa, for example the genera andspecies Musa nana, Musa acuminata, Musa paradisiaca, Musa spp. [banana],Onagraceae, such as the genera Camissonia, Oenothera, for example thegenera and species Oenothera biennis or Camissonia brevipes [eveningprimrose], Palmae, such as the genus Elacis, for example the genus andspecies Elaeis guineensis [oil palm], Papaveraceae, such as the genusPapaver, for example the genera and species Papaver orientale, Papaverrhoeas, Papaver dubium [poppy], Pedaliaceae, such as the genus Sesamum,for example the genus and species Sesamum indicum [sesame], Piperaceae,such as the genera Piper, Artanthe, Peperomia, Steffensia, for examplethe genera and species Piper aduncum, Piper amalego, Piperangustifolium, Piper auritum, Piper betel, Piper cubeba, Piper longum,Piper nigrum, Piper retrofractum, Artanthe adunca, Artanthe elongate,Peperomia elongate, Piper elongatum, Steffensia elongate [cayennepepper], Poaceae, such as the genera Hordeum, Secale, Avena, Sorghum,Andropogon, Holcus, Panicum, Oryza, Zea (maize), Triticum, for examplethe genera and species Hordeum vulgare, Hordeum jubatum, Hordeummurinum, Hordeum secalinum, Hordeum distichon, Hordeum aegiceras,Hordeum hexastichon, Hordeum hexastichum, Hordeum irregulare, Hordeumsativum, Hordeum secalinum [barley], Secale cereale [rye], Avena Avenafatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida [oats],Sorghum bicolor, Sorghum halepense, Sorghum saccharatum, Sorghumvulgare, Andropogon drummondii, Holcus bicolor, Holcus sorghum, Sorghumaethiopicum, Sorghum arundinaceum, Sorghum caffrorum, Sorghum cernuum,Sorghum dochna, Sorghum drummondii, Sorghum durra, Sorghum guineense,Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum, Sorghumsubglabrescens, Sorghum verticilliflorum, Sorghum vulgare, Holcushalepensis, Sorghum miliaceum, Panicum militaceum [millet], Oryzasativa, Oryza latifolia [rice], Zea mays [maize], Triticum aestivum,Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha,Triticum sativum or Triticum vulgare [wheat], Porphyridiaceae, such asthe genera Chroothece, Flintiella, Petrovanella, Porphyridium, Rhodella,Rhodosorus, Vanhoeffenia, for example the genus and species Porphyridiumcruentum, Proteaceae, such as the genus Macadamia, for example the genusand species Macadamia intergrifolia [macadamia], Prasinophyceae such asthe genera Nephroselmis, Prasinococcus, Scherffelia, Tetraselmis,Mantoniella, Ostreococcus, for example the genera and speciesNephroselmis olivacea, Prasinococcus capsulatus, Scherffelia dubia,Tetraselmis chuff, Tetraselmis suecica, Mantoniella squamata,Ostreococcus tauri, Rubiaceae such as the genus Cofea, for example thegenera and species Cofea spp., Coffea arabica, Coffea canephora orCoffea liberica [coffee], Scrophulariaceae such as the genus Verbascum,for example the genera and species Verbascum blattaria, Verbascumchaixii, Verbascum densiflorum, Verbascum lagurus, Verbascumlongifolium, Verbascum lychnitis, Verbascum nigrum, Verbascum olympicum,Verbascum phlomoides, Verbascum phoenicum, Verbascum pulverulentum orVerbascum thapsus [mullein], Solanaceae such as the genera Capsicum,Nicotiana, Solanum, Lycopersicon, for example the genera and speciesCapsicum annuum, Capsicum annuum var. glabriusculum, Capsicum frutescens[pepper], Capsicum annuum [paprika], Nicotiana tabacum, Nicotiana alata,Nicotiana attenuata, Nicotiana glauca, Nicotiana langsdorffii, Nicotianaobtusifolia, Nicotiana quadrivalvis, Nicotiana repanda, Nicotianarustica, Nicotiana sylvestris [tobacco], Solanum tuberosum [potato],Solanum melongena [eggplant], Lycopersicon esculentum, Lycopersiconlycopersicum, Lycopersicon pyriforme, Solanum integrifolium or Solanumlycopersicum [tomato], Sterculiaceae, such as the genus Theobroma, forexample the genus and species Theobroma cacao [cacao] or Theaceae, suchas the genus Camellia, for example the genus and species Camelliasinensis [tea]. In particular preferred plants to be used as transgenicplants in accordance with the present invention are oil fruit cropswhich comprise large amounts of lipid compounds, such as peanut, oilseedrape, canola, sunflower, safflower, poppy, mustard, hemp, castor-oilplant, olive, sesame, Calendula, Punica, evening primrose, mullein,thistle, wild roses, hazelnut, almond, macadamia, avocado, bay,pumpkin/squash, linseed, soybean, pistachios, borage, trees (oil palm,coconut, walnut) or crops such as maize, wheat, rye, oats, triticale,rice, barley, cotton, cassava, pepper, Tagetes, Solanaceae plants suchas potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa orbushy plants (coffee, cacao, tea), Salix species, and perennial grassesand fodder crops. Preferred plants according to the invention are oilcrop plants such as peanut, oilseed rape, canola, sunflower, safflower,poppy, mustard, hemp, castor-oil plant, olive, Calendula, Punica,evening primrose, pumpkin/squash, linseed, soybean, borage, trees (oilpalm, coconut). Especially preferred are plants which are high in C18:2-and/or C18:3-fatty acids, such as sunflower, safflower, tobacco,mullein, sesame, cotton, pumpkin/squash, poppy, evening primrose,walnut, linseed, hemp, thistle or safflower. Very especially preferredplants are plants such as safflower, sunflower, poppy, evening primrose,walnut, linseed, or hemp.

Preferred mosses are Physcomitrella or Ceratodon. Preferred algae arelsochrysis, Mantoniella, Ostreococcus or Crypthecodinium, andalgae/diatoms such as Phaeodactylum or Thraustochytrium. Morepreferably, said algae or mosses are selected from the group consistingof: Shewanella, Physcomitrella, Thraustochytrium, Fusarium,Phytophthora, Ceratodon, lsochrysis, Aleurita, Muscarioides,Mortierella, Phaeodactylum, Cryphthecodinium, specifically from thegenera and species Thallasiosira pseudonona, Euglena gracilis,Physcomitrella patens, Phytophtora infestans, Fusarium graminaeum,Cryptocodinium cohnii, Ceratodon purpureus, lsochrysis galbana, Aleuritafarinosa, Thraustochytrium sp., Muscarioides viallii, Mortierellaalpina, Phaeodactylum tricornutum or Caenorhabditis elegans orespecially advantageously Phytophtora infestans, Thallasiosirapseudonona and Cryptocodinium cohnii.

Transgenic plants may be obtained by transformation techniques aspublished, and cited, in: Plant Molecular Biology and Biotechnology (CRCPress, Boca Raton, Fla.), chapter 6/7, pp. 71-119 (1993); F. F. White,Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, vol.1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press,1993, 15-38; B. Jenes et al., Techniques for Gene Transfer, in:Transgenic Plants, vol. 1, Engineering and Utilization, Ed.: Kung and R.Wu, Academic Press (1993), 128-143; Potrykus, Annu. Rev. Plant Physiol.Plant Molec. Biol. 42 (1991), 205-225. Preferably, transgenic plants canbe obtained by T-DNA-mediated transformation. Such vector systems are,as a rule, characterized in that they contain at least the vir genes,which are required for the Agrobacterium-mediated transformation, andthe sequences which delimit the T-DNA (T-DNA border). Suitable vectorsare described elsewhere in the specification in detail.

Preferably, a multicellular micro-organism as used herein refers toprotists or diatoms. More preferably, it is selected from the group ofthe families Dinophyceae, Turaniellidae or Oxytrichidae, such as thegenera and species: Crypthecodinium cohnii, Phaeodactylum tricornutum,Stylonychia mytilus, Stylonychia pustulate, Stylonychia putrina,Stylonychia notophora, Stylonychia sp., Colpidium campylum or Colpidiumsp.

The present invention further encompasses a method for the manufactureof a compound having a structure as shown in the general formula I

wherein the variables and substituents in formula I are

-   -   R¹=hydroxyl, coenzyme A (thioester), lysophosphatidylcholine,        lysophosphatidylethanolamine, lysophosphatidylglycerol,        lysodiphosphatidylglycerol, lysophosphatidylserine,        lysophosphatidylinositol, sphingo base or a radical of the        formula II

-   -   R²=hydrogen, lysophosphatidylcholine,        lysophosphatidylethanolamine, lysophosphatidylglycerol,        lysodiphosphatidylglycerol, lysophosphatidylserine,        lysophosphatidylinositol or saturated or unsaturated        C₂-C₂₄-alkylcarbonyl,    -   R³=hydrogen, saturated or unsaturated C₂-C₂₄-alkylcarbonyl, or        R² and R³ independently of each other are a radical of the        formula Ia:

-   -   n=2, 3, 4, 5, 6, 7 or 9, m=2, 3, 4, 5 or 6 and p=0 or 3;        and

wherein said method comprises cultivating (i) the host cell of any ofclaims 3 to 5, (ii) the transgenic non-human organism of claim 12 or 13or (iii) a host cell or a transgenic non-human organism comprising apolynucleotide comprising a nucleic acid sequence as shown in any one ofSEQ ID NOs: 6, 7, 9, 11, 13, 30, 33 or 35 or which encodes a polypeptidehaving an amino acid sequence as shown in any one of SEQ ID NOs: 8, 10,12, 14, 31, 34 or 36 under conditions which allow biosynthesis of thesaid compound, preferably, with a content of at least 1% by weight ofthese compounds based on the total lipid content of the host cell or thetransgenic non-human organism.

Preferably, R¹ in the general formula I is hydroxyl, coenzyme A(thioester), ysophosphatidylcholine, lysophosphatidylethanolamine,lysophosphatidylglycerol, lysodiphosphatidylglycerol,lysophosphatidylserine, lysophosphatidylinositol, sphingo base or aradical of the formula II

The abovementioned radicals of R¹ are always bonded to the compounds ofthe general formula I in the form of their thioesters.

Preferably, R² in the general formula II is hydrogen,lysophosphatidylcholine, lysophosphatidylethanolamine,lysophosphatidylglycerol, lysodiphosphatidylglycerol,lysophosphatidylserine, lysophosphatidylinositol or saturated orunsaturated C₂-C₂₄-alkylcarbonyl. Moreover, alkyl radicals which may bementioned are substituted or unsubstituted, saturated or unsaturatedC₂-C₂₄-alkylcarbonyl chains such as ethylcarbonyl, n-propylcarbonyl,n-butylcarbonyl, n-pentylcarbonyl, n-hexylcarbonyl, n-heptylcarbonyl,n-octylcarbonyl, n-nonylcarbonyl, n-decylcarbonyl, n-undecylcarbonyl,n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl,n-pentadecylcarbonyl, n-hexadecylcarbonyl, n-heptadecylcarbonyl,n-octadecylcarbonyl-, n-nonadecylcarbonyl, n-eicosylcarbonyl,n-docosanylcarbonyl- or n-tetracosanylcarbonyl, which comprise one ormore double bonds. Saturated or unsaturated C₁₀-C₂₂-alkylcarbonylradicals such as n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl,n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl,n-hexadecylcarbonyl, n-heptadecylcarbonyl, n-octadecylcarbonyl,n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl orn-tetracosanylcarbonyl, which comprise one or more double bonds, arepreferred. Preferred are saturated and/or unsaturatedC₁₀-C₂₂-alkylcarbonyl radicals such as C₁₀-alkylcarbonyl,C₁₁-alkylcarbonyl, C₁₂-alkylcarbonyl, C₁₃-alkylcarbonyl,C₁₄-alkylcarbonyl, C₁₆-alkylcarbonyl, C₁₈-alkylcarbonyl,C₂₀-alkylcarbonyl or C₂₂-alkylcarbonyl radicals which comprise one ormore double bonds. Particularly preferred are saturated or unsaturatedC₂₀-C₂₂-alkylcarbonyl radicals such as C₂₀-alkylcarbonyl orC₂₂-alkylcarbonyl radicals which comprise one or more double bonds.These preferred radicals can comprise two, three, four, five or sixdouble bonds.

The particularly preferred radicals with 20 or 22 carbon atoms in thefatty acid chain comprise up to six double bonds, advantageously two,three, four or five double bonds, especially preferably two, three orfour double bonds. All the abovementioned radicals are derived from thecorresponding fatty acids.

Preferably, R³ in the formula II is hydrogen, saturated or unsaturatedC₂-C₂₄-alkylcarbonyl. Alkyl radicals which may be mentioned aresubstituted or unsubstituted, saturated or unsaturatedC₂-C₂₄-alkylcarbonyl chains such as ethylcarbonyl, n-propylcarbonyl,n-butylcarbonyl-, n-pentylcarbonyl, n-hexylcarbonyl, n-heptylcarbonyl,n-octylcarbonyl, n-nonylcarbonyl, n-decylcarbonyl, n-undecylcarbonyl,n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl,n-pentadecylcarbonyl, n-hexadecylcarbonyl, n-heptadecylcarbonyl,n-octadecylcarbonyl-, n-nonadecylcarbonyl, n-eicosylcarbonyl,n-docosanylcarbonyl- or n-tetracosanylcarbonyl, which comprise one ormore double bonds. Saturated or unsaturated C₁₀-C₂₂-alkylcarbonylradicals such as n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl,n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl,n-hexadecylcarbonyl, n-heptadecylcarbonyl, n-octadecylcarbonyl,n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl orn-tetracosanylcarbonyl, which comprise one or more double bonds, arepreferred. Preferred are saturated and/or unsaturatedC₁₀-C₂₂-alkylcarbonyl radicals such as C₁₀-alkylcarbonyl,C₁₁-alkylcarbonyl, C₁₂-alkylcarbonyl, C₁₃-alkylcarbonyl,C₁₄-alkylcarbonyl, C₁₆-alkylcarbonyl, C₁₈-alkylcarbonyl,C₂₀-alkylcarbonyl or C₂₂-alkylcarbonyl radicals which comprise one ormore double bonds. Particularly preferred are saturated or unsaturatedC₂₀-C₂₂-alkylcarbonyl radicals such as C₂₀-alkylcarbonyl orC₂₂-alkylcarbonyl radicals which comprise one or more double bonds.These preferred radicals can comprise two, three, four, five or sixdouble bonds. The particularly preferred radicals with 20 or 22 carbonatoms in the fatty acid chain comprise up to six double bonds,advantageously two, three, four or five double bonds, especiallypreferably two, three or four double bonds. All the abovementionedradicals are derived from the corresponding fatty acids.

The abovementioned radicals of R¹, R² and R³ can be substituted byhydroxyl and/or epoxy groups and/or can comprise triple bonds.

The polyunsaturated fatty acids produced in the process according to theinvention advantageously comprise at least two, advantageously three,four, five or six, double bonds. The fatty acids especiallyadvantageously comprise two, three, four or five double bonds. Fattyacids produced in the method of the present invention, preferably,comprise 20 or 22 carbon atoms in the fatty acid chain. Saturated fattyacids are advantageously reacted to a minor degree, or not at all, bythe nucleic acids used in the process. To a minor degree is to beunderstood as meaning that the saturated fatty acids are reacted withless than 5% of the activity, advantageously less than 3%, especiallyadvantageously with less than 2% of the activity in comparison withpolyunsaturated fatty acids. These fatty acids which have been producedcan be produced in the process as a single product or be present in afatty acid mixture.

Advantageously, the substituents R² or R³ in the general formulae I andII independently of one another are saturated or unsaturatedC₂₀-C₂₂-alkylcarbonyl; especially advantageously, are independently ofone another unsaturated C₂₀- or C₂₂-alkylcarbonyl with at least twodouble bonds.

The polyunsaturated fatty acids produced by the method of the presentinvention are, preferably, bound in membrane lipids and/ortriacylglycerides, but may also occur in the organisms as free fattyacids or else bound in the form of other fatty acid esters. In thiscontext, they may be present as “pure products” or else advantageouslyin the form of mixtures of various fatty acids or mixtures of differentglycerides. The various fatty acids which are bound in thetriacylglycerides can be derived from short-chain fatty acids with 4 to6 C atoms, medium-chain fatty acids with 8 to 12 C atoms or long-chainfatty acids with 14 to 24 C atoms. In accordance with the method of thepresent invention, preferred are the long-chain fatty acids, especiallythe LCPUFAs of C₂₀- and/or C₂₂-fatty acids.

The method of the invention, advantageously, yields fatty acid esterswith polyunsaturated C₂₀- and/or C₂₂-fatty acid molecules with at leasttwo double bonds in the fatty acid ester, preferably, with at least two,three, four, five or six double bonds in the fatty acid ester, morepreferably, of at least three, four, five or six double bonds in thefatty acid ester. These fatty acid esteres, preferably, lead to thesynthesis of ETA, EPA and/or DHA.

The fatty acid esters with polyunsaturated C₂₀- and/or C₂₂-fatty acidmolecules can be isolated in the form of an oil or lipid, for example,in the form of compounds such as sphingolipids, phosphoglycerides,lipids, glycolipids such as glycosphingolipids, phospholipids such asphosphatidylethanolamine, phosphatidylcholine, phosphatidylserine,phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol,monoacylglycerides, diacylglycerides, triacylglycerides or other fattyacid esters such as the acetylcoenzyme A esters which comprise thepolyunsaturated fatty acids with at least two, three, four, five or six,preferably five or six, double bonds, from the organisms which were usedfor the preparation of the fatty acid esters. Preferably, they areisolated in the form of their diacylglycerides, triacylglycerides and/orin the form of phosphatidylcholine, especially preferably in the form ofthe triacylglycerides. In addition to these esters, the polyunsaturatedfatty acids are also present in the non-human transgenic organisms orhost cells, preferably in the plants, as free fatty acids or bound inother compounds. As a rule, the various abovementioned compounds (fattyacid esters and free fatty acids) are present in the organisms with anapproximate distribution of 80 to 90% by weight of triglycerides, 2 to5% by weight of diglycerides, 5 to 10% by weight of monoglycerides, 1 to5% by weight of free fatty acids, 2 to 8% by weight of phospholipids,the total of the various compounds amounting to 100% by weight.

In the method of the invention, the LCPUFAs which have been produced areproduced in a content of at least 1% by weight, at least 2% by weight,at least 3% by weight, advantageously at least 5% by weight, preferablyat least 8% by weight, especially preferably at least 10% by weight,very especially preferably at least 15% by weight, based on the totalfatty acids in the non-human transgenic organisms or the host cellreferred to above. The fatty acids are, preferably, produced in boundform. It is possible, with the aid of the polynucleotides andpolypeptides of the present invention, for these unsaturated fatty acidsto be positioned at the sn1, sn2 and/or sn3 position of thetriglycerides which are, preferably, to be produced.

In the LCPUFA manufacturing method of the present invention thepolynucleotides and polypeptides of the present invention may be usedwith at least one further polynucleotide encoding an enzyme of the fattyacid or lipid biosynthesis. Preferred enzymes are in this context theΔ4-desaturase, Δ5-desaturase, Δ6-desaturase, Δ8-desaturase, Δ5-elongase,Δ6-elongase and/or Δ9-elongase gene. These enzymes reflect theindividual steps according to which the end products of the method ofthe present invention, for example, ETA, EPA or DHA are produced fromthe starting compounds linoleic acid (C18:2) or linolenic acid (C18:3).As a rule, these compounds are not generated as essentially pureproducts. Rather, small traces of the precursors may be also present inthe end product. If, for example, both linoleic acid and linolenic acidare present in the starting organism, or the starting plant, the endproducts, such as ETA, EPA or DHA, are present as mixtures. Theprecursors should advantageously not amount to more than 20% by weight,preferably not to more than 15% by weight, more preferably, not to morethan 10% by weight, most preferably not to more than 5% by weight, basedon the amount of the end product in question. Advantageously, only STA,only EPA or only, more preferably, DHA, bound or as free acids, areproduced as end products in the process of the invention in a transgenicplant. If the compounds ETA, EPA and DHA are produced simultaneously,they are, preferably, produced in a ratio of at least 1:10:20(DHA:ETA:EPA), more preferably, the ratios are 1:5:10 or 1:2:5 and, mostpreferably, 1:0.1:3.

Fatty acid esters or fatty acid mixtures produced by the invention,preferably, comprise 6 to 15% of palmitic acid, 1 to 6% of stearic acid,7-85% of oleic acid, 0.5 to 8% of vaccenic acid, 0.1 to 1% of arachicacid, 7 to 25% of saturated fatty acids, 8 to 85% of monounsaturatedfatty acids and 60 to 85% of polyunsaturated fatty acids, in each casebased on 100% and on the total fatty acid content of the organisms. DHAas a preferred long chain polyunsaturated fatty acid is present in thefatty acid esters or fatty acid mixtures in a concentration of,preferably, at least 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9 or 1%,based on the total fatty acid content. Moreover, the fatty acid estersor fatty acid mixtures which have been produced by the method of theinvention, preferably, comprise further fatty acids selected from thegroup of the fatty acids erucic acid (13-docosaenoic acid), sterculicacid (9,10-methyleneoctadec-9-enoic acid), malvalic acid(8,9-methyleneheptadec-8-enoic acid), chaulmoogric acid(cyclopentenedodecanoic acid), furan fatty acid(9,12-epoxyoctadeca-9,11-dienoic acid), vernolic acid(9,10-epoxyoctadec-12-enoic acid), tariric acid (6-octadecynoic acid),6-nonadecynoic acid, santalbic acid (t11-octadecen-9-ynoic acid),6,9-octadecenynoic acid, pyrulic acid (t10-heptadecen-8-ynoic acid),crepenyninic acid (9-octadecen-12-ynoic acid), 13,14-dihydrooropheicacid, octadecen-13-ene-9,11-diynoic acid, petroselenic acid(cis-6-octadecenoic acid), 9c,12t-octadecadienoic acid, calendulic acid(8t10t12c-octadecatrienoic acid), catalpic acid(9t11t13c-octadecatrienoic acid), eleostearic acid(9c11t13t-octadecatrienoic acid), jacaric acid(8c10t12c-octadecatrienoic acid), punicic acid(9c11t13c-octadecatrienoic acid), parinaric acid(9c11t13t15c-octadecatetraenoic acid), pinolenic acid(all-cis-5,9,12-octadecatrienoic acid), laballenic acid(5,6-octadecadienallenic acid), ricinoleic acid (12-hydroxyoleic acid)and/or coriolic acid (13-hydroxy-9c,11t-octadecadienoic acid). The fattyacid esters or fatty acid mixtures produced by the method of the presentinvention, preferably, comprise less than 0.1%, based on the total fattyacids, or no butyric acid, no cholesterol, no clupanodonic acid(=docosapentaenoic acid, C22:5^(Δ4,8,12,15,21)) and no nisinic acid(tetracosahexaenoic acid, C23:6^(Δ3,8,12,15,18,21)).

By using the polynucleotides or polypeptides of the present invention inthe aforementioned methods, it is envisaged that the transgenicnon-human organisms or host cells provide an increase in the yield ofthe LCPUFAs of at least 50%, at least 80%, at least 100% or at least150% in comparison with a reference organism or cell (i.e. anon-transgenic or non-modified cell) when compared by means of gaschromatography (GC) analysis; see Examples.

Chemically pure LCPUFAs or fatty acid compositions can also besynthesized by the method described above. To this end, the fatty acidsor the fatty acid compositions are isolated from the non-humantransgenic organism, host cell or culture media of host cells, forexample via extraction, distillation, crystallization, chromatography ora combination of these methods. These chemically pure fatty acids orfatty acid compositions are advantageous for applications in the foodindustry sector, the cosmetic sector and especially the pharmacologicalindustry sector.

Genes encoding further enzymes or proteins involved in the fatty acid orlipid metabolism can be also applied for the method of the presentinvention. Suitable genes are, preferably, selected from the groupconsisting of acyl-CoA dehydrogenase(s), acyl-ACP [=acyl carrierprotein] desaturase(s), acyl-ACP thioesterase(s), fatty acid acyltransferase(s), acyl-CoA: lysophospholipid acyltransferases, fatty acidsynthase(s), fatty acid hydroxylase(s), acetyl-coenzyme Acarboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s),fatty acid acetylenases, lipoxygenases, triacylglycerol lipases,allenoxide synthases, hydroperoxide lyases or fatty acid elongase(s) areadvantageously used in combination with the ω-3-desaturase. Genesselected from the group of the Δ4-desaturases, Δ5-desaturases,Δ6-desaturases, Δ3-desaturases, Δ9-desaturases, Δ12-desaturases,Δ5-elongases, Δ6-elongases or Δ9-elongases are, more preferably, used incombination with the above genes and the polynucleotide of the presentinvention.

The polypeptides of the invention preferentially desaturates C₂₀ andC₂₂-LCPUFAs. Within the non-human transgenic organism or the host cell,these fatty acids are converted to at least 10%, 15%, 20%, 25% or 30%from the existing fatty acid pool to give the corresponding ω-3-fattyacids. Preferred substrates of the ω-3-desaturase according to theinvention are the ω-6-fatty acids bound in phospholipids. Table 1 showsthe preferred substrates (i.e. DGLA, ARA and DPA) and the products (i.e.ETA, EPA and DHA).

Preferably, the LCPUFAs produced by the method of the present inventionare synthesized, depending on the fatty acid present in the non-humantransgenic organism or host cell, which act as starting substance forthe synthesis. Since biosynthetic cascades are involved, the endproducts in question are not present in pure form in the organisms orhost cells. Small amounts of the precursor compounds are alwaysadditionally present in the end product. These small amounts amount toless than 20% by weight, advantageously less than 15% by weight,especially advantageously less than 10% by weight, very especiallyadvantageously less than 5, 4, 3, 2, or 1% by weight, based on the endproducts.

In addition to the synthesis based on endogenous precursors present inthe non-human transgenic organism or host cell, the fatty acids can alsobe fed externally. Preferred substrates in this context aredihomo-γ-linolenic acid (C20:3^(Δ8,11,14)), arachidonic acid(C20:4^(Δ5,8,11,14)), and docosapentaenoic acid (C22:5^(Δ4,7,10,13,15)).

To increase the yield in the above-described method for the productionof oils and/or triglycerides with an advantageously elevated content ofpolyunsaturated fatty acids, it is preferred to increase the amount ofstarting product for the synthesis of fatty acids; this can be achieved,for example, by introducing, into the non-human transgenic organism orhost cell, a nucleic acid which encodes a polypeptide withΔ12-desaturase activity. This is particularly preferred in oil-producingnon-human organisms such as oilseed rape which are high in oleic acid.Since these organisms are only low in linoleic acid (Mikoklajczak etal., Journal of the American Oil Chemical Society, 38, 1961, 678-681),the use of the abovementioned Δ12-desaturases for producing the startingmaterial linoleic acid is advantageous.

In a preferred embodiment of the method of the present invention, thesaid method, furthermore, comprises the step of obtaining the oils,lipids or free fatty acids from the organism or the host cell. It is tobe understood that in case a host cell is exploited as a source, theLCPUFAs to be manufactured can be also obtained form the culture media.

In the case of plant cells, plant tissue or plant organs, “growing” isunderstood as meaning, for example, the cultivation on or in a nutrientmedium, or of the intact plant on or in a substrate, for example in ahydroponic culture, potting compost or on arable land.

Transgenic plants which comprise the polyunsaturated fatty acidssynthesized in the method according to the invention can advantageouslybe marketed directly without there being any need for the oils, lipidsor fatty acids synthesized to be isolated. Plants for the methodaccording to the invention are understood as meaning intact plants andall plant parts, plant organs or plant parts such as leaf, stem, seed,root, tubers, anthers, fibers, root hairs, stalks, embryos, calli,cotelydons, petioles, harvested material, plant tissue, reproductivetissue and cell cultures which are derived from the transgenic plantand/or can be used for bringing about the transgenic plant. In thiscontext, the seed comprises all parts of the seed such as the seedcoats, epidermal cells, seed cells, endosperm or embryonic tissue.However, the compounds produced in the method according to the inventioncan also be isolated from the organisms, advantageously the plants, inthe form of their oils, fat, lipids and/or free fatty acids. LCPUFAsproduced by this method can be harvested by harvesting the organismseither from the culture in which they grow, or from the field. This canbe done via pressing or extraction of the plant parts, preferably theplant seeds. In this context, the oils, fats, lipids and/or free fattyacids can be obtained by what is known as cold-beating or cold-pressingwithout applying heat by pressing. To allow for greater ease ofdisruption of the plant parts, specifically the seeds, they arepreviously comminuted, steamed or roasted. The seeds which have beenpretreated in this manner can subsequently be pressed or extracted withsolvent such as warm hexane. The solvent is subsequently removed again.In the case of microorganisms, for example, these are harvested and thenextracted directly without further processing steps, or else disruptedand then extracted via various methods with which the skilled worker isfamiliar. In this manner, more than 96% of the compounds produced in theprocess can be isolated. Thereafter, the resulting products areprocessed further, i.e. refined. In this process, substances such as theplant mucilages and suspended matter are first removed. What is known asdesliming can be effected enzymatically or, for example,chemico-physically by addition of acid such as phosphoric acid.Thereafter, the free fatty acids are removed by treatment with a base,for example, sodium hydroxide solution. The resulting product is washedthoroughly with water to remove the alkali remaining in the product andthen dried. To remove the pigment remaining in the product, the productsare subjected to bleaching, for example using fuller's earth or activecharcoal. At the end, the product is deodorized, for example usingsteam.

One embodiment of the invention are therefore oils, lipids or fattyacids or fractions thereof which have been prepared by theabove-described process, especially preferably oil, lipid or a fattyacid composition which comprise LCPUFAs and originate from transgenicplants.

As described above, these oils, lipids or fatty acids, preferably,comprise 6 to 15% of palmitic acid, 1 to 6% of stearic acid, 7-85% ofoleic acid, 0.5 to 8% of vaccenic acid, 0.1 to 1% of arachic acid, 7 to25% of saturated fatty acids, 8 to 85% of monounsaturated fatty acidsand 60 to 85% of polyunsaturated fatty acids, in each case based on 100%and on the total fatty acid content of the organisms. Preferred LCPUFAspresent in the fatty acid esters or fatty acid mixtures is, preferably,at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1% of DHA, EPAor ETA, based on the total fatty acid content. Moreover, the fatty acidesters or fatty acid mixtures which have been produced by the process ofthe invention, preferably, comprise further fatty acids selected fromthe group of the fatty acids erucic acid (13-docosaenoic acid),sterculic acid (9,10-methyleneoctadec-9-enoic acid), malvalic acid(8,9-methyleneheptadec-8-enoic acid), chaulmoogric acid(cyclopentenedodecanoic acid), furan fatty acid(9,12-epoxyoctadeca-9,11-dienoic acid), vernonic acid(9,10-epoxyoctadec-12-enoic acid), tariric acid (6-octadecynoic acid),6-nonadecynoic acid, santalbic acid (t11-octadecen-9-ynoic acid),6,9-octadecenynoic acid, pyrulic acid (t10-heptadecen-8-ynoic acid),cre-penyninic acid (9-octadecen-12-ynoic acid), 13,14-dihydrooropheicacid, octadecen-13-ene-9,11-diynoic acid, petroselenic acid(cis-6-octadecenoic acid), 9c,12t-octadecadienoic acid, calendulic acid(8t10t12c-octadecatrienoic acid), catalpic acid(9t11t13c-octadecatrienoic acid), eleostearic acid(9c11t13t-octadecatrienoic acid), jacaric acid(8c10t12c-octadecatrienoic acid), punicic acid(9c11t13c-octadecatrienoic acid), parinaric acid(9c11t13t15c-octadecatetraenoic acid), pinolenic acid(all-cis-5,9,12-octadecatrienoic acid), laballenic acid(5,6-octadecadienallenic acid), ricinoleic acid (12-hydroxyoleic acid)and/or coriolic acid (13-hydroxy-9c,11t-octadecadienoic acid). The fattyacid esters or fatty acid mixtures produced by the process according tothe invention advantageously comprise less than 0.1%, based on the totalfatty acids, or no butter butyric acid, no cholesterol, no clupanodonicacid (=dpcpsapentaenoic acid, C22:5^(Δ4,8,12,15,21)) and no nisinic acid(tetracosahexaenoic acid, C23:6^(Δ3,8,12,15,18,21)).

The oils, lipids or fatty acids according to the invention, preferably,comprise at least 0.5%, 1%, 2%, 3%, 4% or 5%, more preferably, at least6%, 7%, 8%, 9% or 10%, and most preferably at least 11%, 12%, 13%, 14%or 15% of ETA, EPA and/or of DHA, based on the total fatty acid contentof the production organism, advantageously of a plant, especially of anoil crop such as soybean, oilseed rape, coconut, oil palm, safflower,flax, hemp, castor-oil plant, Calendula, peanut, cacao bean, sunfloweror the abovementioned other monocotyledonous or dicotyledonous oilcrops.

A further embodiment according to the invention is the use of the oil,lipid, fatty acids and/or the fatty acid composition in feedstuffs,foodstuffs, dietary supplies, cosmetics or pharmaceutical compositionsas set forth in detail below. The oils, lipids, fatty acids or fattyacid mixtures according to the invention can be used in the manner withwhich the skilled worker is familiar for mixing with other oils, lipids,fatty acids or fatty acid mixtures of animal origin such as, forexample, fish oils.

The terms “oil”, “lipid” or “fat” are understood as meaning a fatty acidmixture comprising unsaturated or saturated, preferably esterified,fatty acid(s). The oil, lipid or fat is preferably high inpolyunsaturated free or, advantageously, esterified fatty acid(s), inparticular the preferred LCPUFAs referred to herein above. The amount ofunsaturated esterified fatty acids preferably amounts to approximately30%, a content of 50% is more preferred, a content of 60%, 70%, 80% ormore is even more preferred. For the analysis, the fatty acid contentcan, for example, be determined by GC after converting the fatty acidsinto the methyl esters by transesterification. The oil, lipid or fat cancomprise various other saturated or unsaturated fatty acids, for examplecalendulic acid, palmitic acid, palmitoleic acid, stearic acid, oleicacid and the like. The content of the various fatty acids in the oil orfat can vary, in particular depending on the starting organism.

The polyunsaturated fatty acids with at least two double bonds, whichacids are produced by the method of the present invention are, asdescribed in detail above. They can be liberated, for example, viatreatment with alkali, for example aqueous KOH or NaOH, or acidhydrolysis, preferably in the presence of an alcohol such as methanol orethanol, or via enzymatic cleavage, and isolated via, for example, phaseseparation and subsequent acidification via, for example, H₂SO₄. Thefatty acids can also be liberated directly without the above-describedprocessing step.

If microorganisms are used as host cells or non-human transgenicorganisms in the method of the present invention, they will be cultured,or grown, in the manner with which the skilled worker is familiar,depending on the microorganism to be used. As a rule, microorganismswill be grown in a liquid medium comprising a carbon source, mostly inthe form of sugars, a nitrogen source, mostly in the form of organicnitrogen sources such as yeast extract or salts such as ammoniumsulfate, trace elements such as iron, manganese and magnesium salts,and, if appropriate, vitamins, at temperatures between 0° C. and 100°C., preferably between 10° C. to 60° C., while gassing in oxygen. Duringthis process, the pH of the liquid nutrient may be kept constant, i.e.regulated during the culture period, or not. The culture can be effectedbatchwise, semi-batchwise or continuously. Nutrients can be introducedat the beginning of the fermentation or fed in semicontinuously orcontinuously. The polyunsaturated fatty acids produced can be isolatedfrom the organisms by methods with which the skilled worker is familiar,as described above; for example via extraction, distillation,crystallization, if appropriate salt precipitation and/orchromatography. To do so, the organisms can advantageously be disruptedbeforehand.

Culturing of the microorganism may be carried out at a temperature ofbetween 0° C. to 95° C., preferably between 10° C. to 85° C., morepreferably between 15° C. to 75° C., most preferably between 15° C. to45° C.

The pH shall be maintained at between pH 4 and pH 12, preferably betweenpH 6 and pH 9, especially preferably between pH 7 and pH 8.

The process according to the invention can be carried out batchwise,semibatchwise or continuously. A summary of known cultivation methods isto be found in the textbook by Chmiel (Bioprozeβtechnik 1. Einführung indie Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or inthe textbook by Storhas (Bioreaktoren and periphere Einrichtungen(Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

The culture medium to be used must satisfy in a suitable manner thedemands of the respective strains. There are descriptions of culturemedia for various microorganisms in the handbook “Manual of Methods forGeneral Bacteriology” of the American Society for Bacteriology(Washington D.C., USA, 1981).

These media which can be employed according to the invention usuallycomprise one or more carbon sources, nitrogen sources, inorganic salts,vitamins and/or trace elements, as described above.

Preferred carbon sources are sugars such as mono-, di- orpolysaccharides. Examples of very good carbon sources are glucose,fructose, mannose, galactose, ribose, sorbose, ribulose, lactose,maltose, sucrose, raffinose, starch or cellulose. Sugars can be put inthe media also via complex compounds such as molasses, or otherby-products of sugar refining. It may also be advantageous to addmixtures of various carbon sources. Other possible carbon sources areoils and fats such as, for example, soybean oil, sunflower oil, peanutoil and/or coconut fat, fatty acids such as, for example, palmitic acid,stearic acid and/or linoleic acid, alcohols and/or polyalcohols such as,for example, glycerol, methanol and/or ethanol and/or organic acids suchas, for example, acetic acid and/or lactic acid.

Nitrogen sources are usually organic or inorganic nitrogen compounds ormaterials comprising these compounds. Examples of nitrogen sourcesinclude ammonia gas, ammonia liquid or ammonium salts such as ammoniumsulfate, ammonium chloride, ammonium phosphate, ammonium carbonate orammonium nitrate, nitrates, urea, amino acids or complex nitrogensources such as corn steep liquor, soybean flour, soybean protein, yeastextract, meat extract and others. The nitrogen sources may be usedsingly or as mixtures.

Inorganic salt compounds which may be present in the media comprise thechloride, phosphoric or sulfate salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

For producing sulfur-containing fine chemicals, especially methionine,it is possible to use as sulfur source inorganic sulfur-containingcompounds such as, for example, sulfates, sulfites, dithionites,tetrathionates, thiosulfates, sulfides, but also organic sulfurcompounds such as mercaptans and thiols.

It is possible to use as phosphorus source phosphoric acid, potassiumdihydrogen-phosphate or dipotassium hydrogenphosphate or thecorresponding sodium-containing salts.

Chelating agents can be added to the medium in order to keep the metalions in solution. Particularly suitable chelating agents comprisedihydroxyphenols such as catechol or protocatechuate, or organic acidssuch as citric acid.

The fermentation media employed according to the present invention forthe culture of microorganisms normally also comprise other growthfactors such as vitamins or growth promoters, which include for examplebiotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenateand pyridoxine. Growth factors and salts are frequently derived fromcomplex components of the media, such as yeast extract, molasses, cornsteep liquor and the like. Suitable precursors may also be added to theculture medium. The exact composition of the compounds in the mediadepends greatly on the particular experiment and will be decidedindividually for each specific case. Information on optimization ofmedia is obtainable from the textbook “Applied Microbiol. Physiology, APractical Approach” (editors P. M. Rhodes, P. F. Stanbury, IRL Press(1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also bepurchased from commercial suppliers, such as Standard 1 (Merck) or BHI(Brain heart infusion, DIFCO) and the like.

All the components of the media are sterilized either by heat (20 min at1.5 bar and 121° C.) or by filter sterilization. The components can besterilized either together or, if necessary, separately. All thecomponents of the media may be present at the start of culturing oroptionally be added continuously or batchwise.

The temperature of the culture is normally between 15° C. and 45° C.,preferably at 25° C. to 40° C., and can be kept constant or changedduring the experiment. The pH of the medium should be in the range from5 to 8.5, preferably around 7.0. The pH for the culturing can becontrolled during the culturing by adding basic compounds such as sodiumhydroxide, potassium hydroxide, ammonia or aqueous ammonia or acidiccompounds such as phosphoric acid or sulfuric acid. The development offoam can be controlled by employing antifoams such as, for example,fatty acid polyglycol esters. The stability of plasmids can bemaintained by adding to the medium suitable substances with a selectiveaction, such as, for example, antibiotics. Aerobic conditions aremaintained by introducing oxygen or oxygen-containing gas mixtures suchas, for example, ambient air into the culture. The temperature of theculture is normally 20° C. to 45° C., and preferably 25° C. to 40° C.The culture is continued until formation of the desired product is at amaximum. This aim is normally reached within 10 hours to 160 hours.

The dry matter content of the fermentation broths obtained in this wayand comprising in particular polyunsaturated fatty acids is normallyfrom 7.5 to 25% by weight.

The fermentation broth can then be processed further. Depending on therequirement, the biomass can be removed wholly or partly from thefermentation broth by separation methods such as, for example,centrifugation, filtration, decantation or a combination of thesemethods, or left completely in it. The biomass is advantageously workedup after removal.

However, the fermentation broth can also be thickened or concentrated byknown methods such as, for example, with the aid of a rotary evaporator,thin-film evaporator, falling-film evaporator, by reverse osmosis or bynanofiltration, without involving a cell removal step. This concentratedfermentation broth can then be worked up to obtain the fatty acidscomprised therein.

The present invention, furthermore, relates to a method for themanufacture of an oil-, fatty acid- or lipid-containing compositioncomprising the steps of the method of the present invention and thefurther step or formulating the compound as an oil-, fatty acid- orlipid-containing composition.

The term “composition” refers to any composition formulated in solid,liquid or gaseous form. Said composition comprises the compound of theinvention optionally together with suitable auxiliary compounds such asdiluents or carriers or further ingredients. In this context, it isdistinguished for the present invention between auxiliary compounds,i.e. compounds which do not contribute to the effects elicited by thecompounds of the present invention upon application of the compositionfor its desired purpose, and further ingredients, i.e. compounds whichcontribute a further effect or modulate the effect of the compounds ofthe present invention. Suitable diluents and/or carriers depend on thepurpose for which the composition is to be used and the otheringredients. The person skilled in the art can determine such suitablediluents and/or carriers without further ado. Examples of suitablecarriers and/or diluents are well known in the art and include salinesolutions such as buffers, water, emulsions, such as oil/wateremulsions, various types of wetting agents, etc.

In a more preferred embodiment of the oil-, fatty acid orlipid-containing composition, the said composition is further formulatedas a pharmaceutical composition, a cosmetic composition, a foodstuff, afeedstuff, preferably, fish feed or a dietary supply.

The term “pharmaceutical composition” as used herein comprises thecompounds of the present invention and optionally one or morepharmaceutically acceptable carrier. The compounds of the presentinvention can be formulated as pharmaceutically acceptable salts.Acceptable salts comprise acetate, methylester, HCl, sulfate, chlorideand the like. The pharmaceutical compositions are, preferably,administered topically or systemically. Suitable routes ofadministration conventionally used for drug administration are oral,intravenous, or parenteral administration as well as inhalation.However, depending on the nature and mode of action of a compound, thepharmaceutical compositions may be administered by other routes as well.For example, polynucleotide compounds may be administered in a genetherapy approach by using viral vectors or viruses or liposomes.

Moreover, the compounds can be administered in combination with otherdrugs either in a common pharmaceutical composition or as separatedpharmaceutical compositions wherein said separated pharmaceuticalcompositions may be provided in form of a kit of parts.

The compounds are, preferably, administered in conventional dosage formsprepared by combining the drugs with standard pharmaceutical carriersaccording to conventional procedures. These procedures may involvemixing, granulating and compressing or dissolving the ingredients asappropriate to the desired preparation. It will be appreciated that theform and character of the pharmaceutically acceptable carrier or diluentis dictated by the amount of active ingredient with which it is to becombined, the route of administration and other well-known variables.

The carrier(s) must be acceptable in the sense of being compatible withthe other ingredients of the formulation and being not deleterious tothe recipient thereof. The pharmaceutical carrier employed may be, forexample, either a solid, a gel or a liquid. Exemplary of solid carriersare lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia,magnesium stearate, stearic acid and the like. Exemplary of liquidcarriers are phosphate buffered saline solution, syrup, oil such aspeanut oil and olive oil, water, emulsions, various types of wettingagents, sterile solutions and the like. Similarly, the carrier ordiluent may include time delay material well known to the art, such asglyceryl mono-stearate or glyceryl distearate alone or with a wax. Saidsuitable carriers comprise those mentioned above and others well knownin the art, see, e.g., Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa.

The diluent(s) is/are selected so as not to affect the biologicalactivity of the combination. Examples of such diluents are distilledwater, physiological saline, Ringer's solutions, dextrose solution, andHank's solution. In addition, the pharmaceutical composition orformulation may also include other carriers, adjuvants, or nontoxic,nontherapeutic, nonimmunogenic stabilizers and the like.

A therapeutically effective dose refers to an amount of the compounds tobe used in a pharmaceutical composition of the present invention whichprevents, ameliorates or treats the symptoms accompanying a disease orcondition referred to in this specification. Therapeutic efficacy andtoxicity of such compounds can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED50 (thedose therapeutically effective in 50% of the population) and LD50 (thedose lethal to 50% of the population). The dose ratio betweentherapeutic and toxic effects is the therapeutic index, and it can beexpressed as the ratio, LD50/ED50.

The dosage regimen will be determined by the attending physician andother clinical factors; preferably in accordance with any one of theabove described methods. As is well known in the medical arts, dosagesfor any one patient depends upon many factors, including the patient'ssize, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Progress can be monitoredby periodic assessment. A typical dose can be, for example, in the rangeof 1 to 1000 μg; however, doses below or above this exemplary range areenvisioned, especially considering the aforementioned factors.Generally, the regimen as a regular administration of the pharmaceuticalcomposition should be in the range of 1 μg to 10 mg units per day. Ifthe regimen is a continuous infusion, it should also be in the range of1 μg to 10 mg units per kilogram of body weight per minute,respectively. Progress can be monitored by periodic assessment. However,depending on the subject and the mode of administration, the quantity ofsubstance administration may vary over a wide range.

The pharmaceutical compositions and formulations referred to herein areadministered at least once in order to treat or ameliorate or prevent adisease or condition recited in this specification. However, the saidpharmaceutical compositions may be administered more than one time, forexample from one to four times daily up to a non-limited number of days.

Specific pharmaceutical compositions are prepared in a manner well knownin the pharmaceutical art and comprise at least one active compoundreferred to herein above in admixture or otherwise associated with apharmaceutically acceptable carrier or diluent. For making thosespecific pharmaceutical compositions, the active compound(s) willusually be mixed with a carrier or the diluent, or enclosed orencapsulated in a capsule, sachet, cachet, paper or other suitablecontainers or vehicles. The resulting formulations are to be adopted tothe mode of administration, i.e. in the forms of tablets, capsules,suppositories, solutions, suspensions or the like. Dosagerecommendations shall be indicated in the prescribers or usersinstructions in order to anticipate dose adjustments depending on theconsidered recipient.

The term “cosmetic composition” relates to a composition which can beformulated as described for a pharmaceutical composition above. For acosmetic composition, likewise, it is envisaged that the compounds ofthe present invention are also, preferably, used in substantially pureform. Impurities, however, may be less critical than for apharmaceutical composition. Cosmetic compositions are, preferably, to beapplied topically. Preferred cosmetic compositions comprising thecompounds of the present invention can be formulated as a hair tonic, ahair restorer composition, a shampoo, a powder, a jelly, a hair rinse,an ointment, a hair lotion, a paste, a hair cream, a hair spray and/or ahair aerosol.

Finally, as is evident from the above, the present invention, inprinciple, relates to the use of the polynucleotides, vectors, hostcells or transgenic non-human organisms of the present invention for themanufacture of a oil-, fatty acid- or lipid-containing composition.Preferably, the said composition is to be used as a pharmaceuticalcomposition, cosmetic composition, foodstuff, feedstuff, preferably,fish feed or dietary supply.

All references cited in this specification are herewith incorporated byreference with respect to their entire disclosure content and thedisclosure content specifically mentioned in this specification.

The figures show:

FIG. 1: The figure shows a comparison of the DNA sequences for the twoω-3 desaturase polynucleotides from Pythium irrgulare (SEQ ID NOS: 1 and23).

FIG. 2: The figure shows an alignment of the deduced amino acids for thetwo ω-3 desaturase polynucleotides from Pythium irrgulare (SEQ ID NOS: 2and 24).

FIG. 3: A comparison of the deduced amino acids for the ω-3 desaturasepolypeptides from Pythium irrgulare, Phytophthora infestans andSaprolegnia decline is shown.

FIG. 4: GC analysis of fatty acid methyl esters from the yeasttransformant pYES2-O3 and the control pYES2 fed with GDLA.

FIG. 5: GC analysis of fatty acid methyl esters from the yeasttransformant pYES2-O3 and the control pYES2 fed with ARA.

FIG. 6: GC analysis of fatty acid methyl esters from the yeasttransformant pYES2-O3 and the control pYES2 fed with DPA (ω-6).

The following Examples shall merely illustrate the invention. They shallnot be construed, whatsoever, to limit the scope of the invention.

EXAMPLE 1 Isolation of Novel ω-3 Desaturase Polynucleotides From Pythiumirregulare

ω-3 desaturases are the enzymes which are able to convert ω-6 fattyacids into their corresponding ω-3 PUFAs. In order to isolatepolynucleotides encoding said enzymes, Pythium irregulare strain 10951was ordered from ATCC. It was grown in liquid media YETG at roomtemperature for 5 days with constant agitation at 250 rpm. Total RNA wasisolated from the harvested mycelia using TRIzol reagent (Invitrogen).The cDNA was synthesized using the Superscript III first strand kit(Invitrogen). Two pairs of degenerate primers were designed based on theconserved domains of omega-3 desaturase genes. RT-PCR was conducted toamplify the ω-3 fragments using the Pythium cDNA as the template by

TTYTGGGGNTTYTTYACNGT (forward primer; SEQ ID NO: 46) andCCYTTNACYTANGTCCACT. (reverse primer; SEQ ID NO: 47)

A 500 base-pair (bp) fragment was amplified and cloned into pCR4-TOPOvector (Invitrogen). A blast search from the sequence of the 500 byfragment confirmed that it was an omega-3 desaturase gene from Pythiumirregulare.

Based on the sequence of the ω-3 desaturase fragment from Pythiumirregulare, two pairs of race primers and one pair of nested PCR primerswere designed

(TCGCGCTCGCATGTGCTCAACTTCAG, SEQ ID NO: 48 RACE-F1,;TGGTGAC-CACGAGCATCGTGGCGAAG, SEQ ID NO: 49 RACE-R1,;TCCTCACGCCGTTCGAGTCCTGGAAG, SEQ ID NO: 50 RACE-F1,;ATGGTCGTGAAGCCCAAGACGAAGGTC, SEQ ID NO: 51) RACE-R2,.

A Marathon RACE cDNA library (BD Biosciences) was made using themessenger RNA isolated from total RNA from Pythium irregulare. PCRreactions for 3′ and 5′ races were applied to amplify a 800 by and a1000 by fragments, respectively, from 3′ and 5′ RACE. These fragmentswere cloned into pCR4-TOPO vector (Invitrogen). Four positive clonesfrom each race were sequenced and there are some variations among them.Therefore Pythium irregulare may have more than one ω-3 desaturasegenes.

The assembled ω-3 desaturase gene contains a 1092 by of open readingframe. Based the assembled ω-3 desaturase gene, one pair of primers

(TCCGCTCGCCATGGCGTCCAC, O3-Yes1, SEQ ID NO: 52 andTGACCGAT-CACTTAGCTGCAGCTTA, O3-Yes2, SEQ ID NO: 53)was designed to amplify the full length of O3 genes (ω-3 desaturasegenes) from Pythium. The full length O3 from Pythium was cloned intoyeast expression vector pYES2.1/V5-His-TOPO. Eight of full length cloneswere sequenced. Six of them are identical. This gene was designated asO3-Pythiyml. Two of other ones are identical, which was designated asO3-Pythgium2. Two genes are 99% identical (FIG. 1) and they only haveone amino acid different (FIG. 2). The O3 desaturase protein fromPythium is 69% and 60% identical to ω-3 desaturase from P. infestans (WO2005/083053) and ω-3 desaturase gene from Saprolegnia diclina (WO2004/071467) (FIG. 3), respectively. It has low identities to delta-12and delta-15 desaturase genes.

EXAMPLE 2 Characterization of Novel ω-3 Desaturases from Pythiumirregulare

The plasmids containing the full length O3 genes in the yeast expressionvector pYES2.1/V5-His-TOPO were transformed into yeast S. cerevisiae.The positive transformants were selected for uracil auxotrophy on DOB-Uagar plates. To characterize the ω-3 desaturase enzyme activity,positive clones and the control (yeast with pYES2.1 vector) werecultured overnight in DOB-U liquid medium at 28° C. and then grown ininduction medium (DOB-U+Gal+Raf) containing 100 μM of variousexogenously supplied fatty acid substrates at 16° C. for 4 days. Thewhole yeast cells expressing Pythium ω-3 genes were harvested bycentrifugation and washed twice with distilled water. Then the yeastcells were directly transmethylated with methanolic HCl (3N) at 80° C.for 1 hour. The resultant methyl esters were extracted with hexane andanalyzed by gas chromatography (GC). GC was carried out as described inWO 2005/083053.

The expression results showed that ω-3 desaturase from Pythium is notable to desaturase the 18-carbon ω-6 fatty acids, such as LA and GLA. Itdesaturates the ω-6 fatty acids longer than 18-carbon chains, such asDGLA (FIG. 4), ARA (FIG. 5) and DPA (FIG. 6). However, it is morespecific to ARA with over 40% conversion rate (Table 1).

TABLE 1 Production of ω-3 fatty acids from exogenous ω-6 fatty acids inthe yeast transformant (pYES2-O3) and the control yeast pYES2 SubstrateSubstrate (%) Product Product (%) Conversion (%) pYES2 LA 24.50 ALA 0GLA 21.97 SDA 0 DGLA 15.79 ETA 0 ARA 6.45 EPA 0 DPA 8.26 DHA 0.03pYES2-O3 LA 25.56 ALA 0 0 GLA 22.79 SDA 0 0 DGLA 17.78 ETA 1.90 9.65%ARA 7.19 EPA 4.95 40.77% DPA 9.52 DHA 0.20 2.01%

In summary, two ω-3 desaturase isoforms were isolated from Pythiumirregulare and both are able to introduce an ω-3 double bond into ω-6fatty acids longer than 18 carbon chains supplied exogenously in yeast.Moreover, this is apparently the first ω-3 desaturase that is able toconvert the ω-6 DPA into DHA.

1.-18. (canceled)
 19. A polynucleotide comprising a nucleic acidsequences selected from the group consisting of: (a) a nucleic acidsequence as shown in SEQ ID NO: 1 or 23; (b) a nucleic acid sequenceencoding a polypeptide having an amino acid sequence as shown in SEQ IDNO: 2 or 24; (c) a nucleic acid sequence which is at least 70% identicalto the nucleic acid sequence of (a) or (b), wherein said nucleic acidsequence encodes a polypeptide having omega-3 desaturase activity; (d) anucleic acid sequence being a fragment of any one of (a) to (c), whereinsaid fragment encodes a polypeptide having omega-3 desaturase activity;and (e) a nucleic acid sequence encoding a polypeptide having ω-3desaturase activity, wherein said polypeptide comprises a polypeptidepattern as shown in a sequence selected from the group consisting of SEQID NO: 15, 16, 17, 18, 19, 20, 21, 22, 37, 38, 39, 40, 41, 42, 43, 44and
 45. 20. The polynucleotide of claim 19, wherein said polynucleotideis DNA or RNA.
 21. A vector comprising the polynucleotide of claim 19.22. The vector of claim 21, wherein said vector is an expression vector.23. The vector of claim 21, wherein said vector comprises at least onepolynucleotide encoding a further enzyme being involved in thebiosynthesis of fatty acids or lipids.
 24. The vector of claim 23,wherein said further enzyme is selected from the groups consisting of:acyl-CoA dehydrogenase(s), acyl-ACP [=acyl carrier protein]desaturase(s), acyl-ACP thioesterase(s), fatty acid acyltransferase(s),acyl-CoA:lysophospholipid acyltransferase(s), fatty acid synthase(s),fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s),acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acidacetylenase(s), lipoxygenase(s), triacylglycerol lipase(s), allenoxidesynthase(s), hydroperoxide lyase(s) or fatty acid elongase(s),acyl-CoA:lysophospholipid acyltransferase, Δ4-desaturase, Δ5-desaturase,Δ6-desaturase, Δ8-desaturase, Δ9-desaturase, Δ12-desaturase,Δ5-elongase, Δ6-elongase, and Δ9-elongase.
 25. A host cell comprisingthe polynucleotide of claim
 19. 26. The host cell of claim 25, whereinsaid host cell additionally comprises at least one further enzyme beinginvolved in the biosynthesis of fatty acids or lipids.
 27. The host cellof claim 26, wherein said further enzyme is selected from the groupsconsisting of: acyl-CoA dehydrogenase(s), acyl-ACP [=acyl carrierprotein] desaturase(s), acyl-ACP thioesterase(s), fatty acidacyltransferase(s), acyl-CoA:lysophospholipid acyltransferase(s), fattyacid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme Acarboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s),fatty acid acetylenase(s), lipoxygenase(s), triacylglycerol lipase(s),allenoxide synthase(s), hydroperoxide lyase(s) or fatty acidelongase(s), acyl-CoA:lysophospholipid acyltransferase, Δ4-desaturase,Δ5-desaturase, Δ6-desaturase, Δ8-desaturase, Δ9-desaturase,Δ12-desaturase, Δ5-elongase, Δ6-elongase, and Δ9-elongase.
 28. A methodfor the manufacture of a polypeptide having ω-3 desaturase activitycomprising: (a) expressing the polynucleotide of claim 19 in a hostcell; and (b) obtaining the polypeptide encoded by said polynucleotidefrom the host cell.
 29. A polypeptide encoded by the polynucleotide ofclaim
 19. 30. An antibody which specifically recognizes the polypeptideof claim
 29. 31. A transgenic non-human organism comprising thepolynucleotide of claim
 19. 32. The transgenic non-human organism ofclaim 31, wherein said organism is an animal, a plant or a multicellularmicro-organism.
 33. A method for the manufacture of a compound having astructure as shown in the general formula I

wherein the variables and substituents in formula I are R¹=hydroxyl,coenzyme A (thioester), lysophosphatidylcholine,lysophosphatidylethanolamine, lysophosphatidyl glycerol,lysodiphosphatidylglycerol, lysophosphatidylserine,lysophosphatidylinositol, sphingo base or a radical of the formula II

R²=hydrogen, lysophosphatidylcholine, lysophosphatidylethanolamine,lysophosphatidylglycerol, lysodiphosphatidylglycerol,lysophosphatidylserine, lysophosphatidylinositol or saturated orunsaturated C₂-C₂₄-alkylcarbonyl, R³=hydrogen, saturated or unsaturatedC₂-C₂₄-alkylealbonyl, or R² and R³ independently of each other are aradical of the formula Ia:

n=2, 3, 4, 5, 6, 7 or 9, m=2, 3, 4, 5 or 6 and p=0 or 3; and whereinsaid method comprises cultivating (i) a host cell comprising thepolynucleotide of claim 19, (ii) a transgenic non-human organismcomprising the polynucleotide, or (iii) a host cell or a transgenicnon-human organism comprising a polynucleotide comprising a nucleic acidsequence as shown in any one of SEQ ID NOs: 6, 7, 9, 11, 13, 30, 33 or35 or which encodes a polypeptide having an amino acid sequence as shownin any one of SEQ ID NOs: 8, 10, 12, 14, 31, 34 or 36 under conditionswhich allow biosynthesis of the said compound.
 34. A method for themanufacture of an oil-, fatty acid- or lipid-containing compositioncomprising the steps of the method of claim 33 and the further step orformulating the compound as an oil-, fatty acid- or lipid-containingcomposition.
 35. The method of claim 34, wherein said oil-, fatty acidor lipid-containing composition is further formulated as apharmaceutical composition, a cosmetic composition, a foodstuff, afeedstuff, a fish feed or a dietary supply.